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A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xenogeneic/serum-free conditions

Open AccessPublished:August 02, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.06.003

      Abstract

      Background aims

      Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by (i) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell expansion; (ii) donor-specific characteristics, including MSC frequency/quality, that decline with disease state and increasing age; and (iii) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which poses safety and consistency issues.

      Methods

      To overcome these limitations while enabling robust MSC production with constant high yield and quality, the authors developed a chemically defined biomimetic surface coating called isoMATRIX (denovoMATRIX GmbH, Dresden, Germany) and tested its performance during isolation of MSCs.

      Results

      The isoMATRIX facilitates the isolation of significantly higher numbers of MSCs in xenogeneic (xeno)/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. The high proliferation rates can be maintained up to 5 passages after isolation and cells even benefit from a switch towards a proliferation-specific MSC matrix (myMATRIX MSC) (denovoMATRIX GmbH, Dresden, Germany).

      Conclusion

      In sum, isoMATRIX promotes enhanced xeno/serum-free and chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

      Key Words

      Introduction

      Adult stem cells, also called somatic stem cells, offer great promise for the treatment of intractable diseases and disorders. They are isolated from perinatal or adult tissues and bear the advantage of restricted lineage potential, which reduces the risk of tumorigenesis compared with pluripotent stem cells [
      • Grompe M.
      Adult versus embryonic stem cells: It's still a tie.
      ,
      • Passier R.
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      Origin and use of embryonic and adult stem cells in differentiation and tissue repair.
      ]. An attractive source of somatic stem cells is mesenchymal stromal cells (MSCs), a population of multipotent progenitor cells capable of differentiating into osteogenic, adipogenic and chondrogenic lineages. Their diverse therapeutic potential, safety and ability to self-renew with high proliferative capacity make them especially valuable for regenerative medicine and cell therapy [
      • Brown C.
      • et al.
      Mesenchymal stem cells: Cell therapy and regeneration potential.
      ]. MSCs can ameliorate diseases, support engraftment and modulate the host immune response, mainly via the secretion of paracrine factors such as cytokines and growth factors or extracellular vesicles [
      • Pittenger M.F.
      • et al.
      Mesenchymal stem cell perspective: cell biology to clinical progress.
      ,
      • Levy O.
      • et al.
      Shattering barriers toward clinically meaningful MSC therapies.
      ]. The success of such innovative cell therapies depends on the availability of stem cell sources and the efficacy of isolation and expansion techniques to yield sufficient amounts of therapeutically active cells.
      The phenotype, stemness and differentiation potential of MSCs can be affected by growth supplements, isolation procedures and culture conditions, which could limit their clinical applicability [
      • Samsonraj R.M.
      • et al.
      Concise Review : Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine.
      ,
      • Suliman S.
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      Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.
      ,
      • Fekete N.
      • et al.
      GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC.
      ]. Thus, identifying optimal conditions for isolation and in vitro expansion of MSCs is crucial for the development of advanced cell therapies. Optimization includes the adjustment of medium composition, culture substrate, cell seeding density, oxygen and carbon dioxide (CO2) concentration, pH and temperature for more defined and reproducible results. Xenogeneic (xeno)/serum-free media are preferably used for MSC isolation and in vitro expansion as a result of their safety, consistency and capacity to support MSC proliferation [
      • Bui H.T.H.
      • Nguyen L.T.
      • Than U.T.T.
      Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.
      ,
      • Swamynathan P.
      • et al.
      Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study.
      ,
      • Laner-Plamberger S.
      • et al.
      Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.
      ,
      • Gottipamula S.
      • Muttigi M.S.
      • Kolkundkar U.
      • Seetharam R.N.
      Serum-free media for the production of human mesenchymal stromal cells: A review.
      ,
      • Bhat S.
      • Viswanathan P.
      • Chandanala S.
      • Prasanna S.J.
      • Seetharam R.N
      Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions.
      ]. These media often lack the adhesion molecules required for the anchorage-dependent MSCs and therefore need to be complemented by extracellular matrix (ECM) proteins such as collagens, fibronectin, vitronectin or laminin. Various research groups have used such ECM proteins in combination with different media and observed an improvement in MSC attachment, migration and survival, leading to enhanced proliferation [
      • Kasten A.
      • et al.
      Guidance of Mesenchymal Stem Cells on Fibronectin Structured Hydrogel Films.
      ,
      • Somaiah C.
      • et al.
      Collagen Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells.
      ,
      • Muraya K.
      • Kawasaki T.
      • Yamamoto T.
      • Akutsu H.
      Enhancement of Cellular Adhesion and Proliferation in Human Mesenchymal Stromal Cells by the Direct Addition of Recombinant Collagen i Peptide to the Culture Medium.
      ,
      • Mittag F.
      • et al.
      Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.
      ]. These proteins can be of either human or animal origin and have the disadvantages of batch-to-batch variability and concerns regarding pathogen contamination and immune response after cell implantation. Hence, the application of such ECM protein coatings for the production of MSCs for clinical practice would require a strict and extensive validation process to reduce the aforementioned risks. To circumvent these additional costs and workload, the use of recombinant proteins or chemically synthesized peptides is recommended.
      In order to facilitate MSC expansion in highly defined culture conditions, the authors recently developed a chemically defined biomatrix for the in vitro culture of MSCs called myMATRIX MSC (denovoMATRIX GmbH, Dresden, Germany), which enables faster and more reliable expansion in serum-free medium compared with serum-containing conditions. This matrix is formed by the interaction of sulfated glycosaminoglycan mimetics with a biofunctional peptide conjugated to a four-arm polyethylene glycol [
      • Wieduwild Robert
      • Wetzel Richard
      • Husman Dejan
      • Bauer Sophie
      • El-Sayed Iman
      • Duin Sarah
      • Murawala Priyanka
      • Thomas Alvin Kuriakose
      • Wobus Manja
      • Bornhäuser Martin
      • Zhang Y
      Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation.
      ]. It promotes cell adhesion and expansion using different media compositions and supports the long-term culture of MSCs in a serum-free medium with robust growth and high viability while maintaining their differentiation and immunomodulatory capacity, characteristic cell morphology and expression of key stemness markers [
      • Thamm K.
      • et al.
      A Novel Synthetic, Xeno-Free Biomimetic Surface for Serum-Free Expansion of Human Mesenchymal Stromal Cells.
      ].
      MSCs can be isolated from various tissues, such as bone marrow, adipose tissue, umbilical cord or deciduous teeth. The number of stem cells varies considerably depending on the tissue of origin. Bone marrow, for example, harbors the lowest amount of MSCs (1–30 cells/mL to 317 400 cells/mL) followed by adipose tissue (4737 cells/mL to 1 550 000 cells/mL) and umbilical cord tissue (10 000 cells/mL to 4 700 000 cells/cm) [
      • Vangsness J.C.T.
      • Sternberg H.
      • Harris L.
      Umbilical Cord Tissue Offers the Greatest Number ofHarvestable Mesenchymal Stem Cells for Researchand Clinical Application: A Literature Review ofDifferent Harvest Sites.
      ]. Comparative studies show that MSCs from bone marrow, adipose tissue and umbilical cord share a common phenotype and differentiation potential as well as the expression of many cell surface markers. However, they noticeably differ in gene expression patterns, proliferation rate and therapeutic efficacy [
      • Kern S.
      • Eichler H.
      • Stoeve J.
      • Klüter H.
      • Bieback K.
      Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood, or Adipose Tissue.
      ,
      • Wagner W.
      • et al.
      Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.
      ,
      • Ribeiro A.
      • et al.
      Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells.
      ].
      Bone marrow-derived MSCs (BM-MSCs), which are still the predominant source for clinical trials [
      • Zhou T.
      • et al.
      Challenges and advances in clinical applications of mesenchymal stromal cells.
      ], can be isolated using density gradient centrifugation to separate mononuclear cells (MNCs) from plasma, erythrocytes and fat [
      • Tuan S.
      • Ho B.
      • Tanavde V.M.
      • Hui J.H.
      • Lee E.H
      Upregulation of Adipogenesis and Chondrogenesis in MSC Serum-Free Culture.
      ,
      • Hoang V.T.
      • et al.
      Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.
      ]. This MNC population contains about 0.001–0.01% MSCs [
      • Pittenger M.F.
      • et al.
      Multilineage potential of adult human mesenchymal stem cells.
      ], a comparably rare population that needs to be captured in the isolation process. Conventionally, MSCs are isolated via selection through non-specific adhesion to plastic cell culture ware in serum-containing medium over several passages. This process lacks directed selectivity, and the isolation/culture conditions are not optimized to replace the in vivo cellular microenvironment. Ultimately, this might reduce isolation efficiency and could lead to the introduction of early cellular heterogeneity. In addition, the transfer of MSCs from their native microenvironment to the nutrient-rich artificial culture conditions induces a metabolic shift from glycolysis toward oxidative phosphorylation, resulting in high metabolic diversity and accelerated senescence [
      • Yuan X.
      • Logan T.M.
      • Ma T.
      Metabolism in human mesenchymal stromal cells: A missing link between HMSC biomanufacturing and therapy?.
      ]. The use of fetal bovine serum introduces undesirable variability due to its poorly defined composition, leading to inconsistent lot-to-lot performance, appreciable risk of contamination and adverse immunological reactions against xeno-components.
      In an attempt to overcome the concerns regarding serum supplementation, Ho et al. [
      • Ho S.T.B.
      • Tanavde V.M.
      • Hui J.H.
      • Lee E.H
      Upregulation of Adipogenesis and Chondrogenesis in MSC Serum-Free Culture.
      ] used a medium that consisted of Dulbecco's Modified Eagle's Medium (DMEM) and 10% KnockOut Serum Replacement with a cocktail of growth factors. Although the exact isolated cell numbers were not stated, they observed cell attachment similar to serum-containing conditions. To eliminate animal-derived components during isolation and ex vivo expansion of human MSCs for clinical applications, human serum and platelet lysate are frequently used as growth supplements and reported to support enhanced proliferation of MSCs [
      • Laner-Plamberger S.
      • et al.
      Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.
      ,
      • Gottipamula S.
      • Muttigi M.S.
      • Kolkundkar U.
      • Seetharam R.N.
      Serum-free media for the production of human mesenchymal stromal cells: A review.
      ,
      • Abdelrazik H.
      • Spaggiari G.M.
      • Chiossone L.
      • Moretta L
      Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T- and NK-cell proliferation and function.
      ]. However, their quality is affected by several factors, such as preparation procedure, donor age or blood profile and heparin levels (in platelet lysate to prevent clotting) [
      • Hemeda H.
      • Kalz J.
      • Walenda G.
      • Lohmann M.
      • Wagner W.
      Heparin concentration is critical for cell culture with human platelet lysate.
      ,
      • Hemeda H.
      • Giebel B.
      • Wagner W.
      Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.
      ]. Furthermore, concerns regarding immunological responses, the transmission of human infections and the availability of large quantities required for clinical applications have been raised. In 2021, Hoang et al. [
      • Hoang V.T.
      • et al.
      Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.
      ] performed a comprehensive study using four different serum- and xeno-free media for the isolation of MSCs from bone marrow, adipose tissue and umbilical cord tissue. They found two conditions (StemMACS XF MSC expansion media kit + CELLstart substrate and MesenCult-ACF + serum-free attachment substrate) that supported the isolation of MSCs from bone marrow aspirates, with an isolation efficiency of about 0.1 MSCs per MNC in passage zero.
      Adipose tissue-derived MSCs are isolated using enzymatic isolation techniques in which tissue samples are incubated in different enzymes (e.g., trypsin, collagenase or dispase) to release cells from their native microenvironment [
      • Oberbauer E.
      • et al.
      Enzymatic and non-enzymatic isolation systems for adipose tissue-derived cells: current state of the art.
      ,
      • Salehinejad P.
      • Moshrefi M.
      • Eslaminejad T.
      An Overview on Mesenchymal Stem Cells Derived from Extraembryonic Tissues: Supplement Sources and Isolation Methods.
      ]. This method not only leads to the destruction of the ECM but can also affect cell surface antigens, membrane integrity and cell viability [
      • Widowati W.
      • et al.
      Comparative Analysis of Wharton’s Jelly Mesenchymal Stem Cell (WJ-MSCs) Isolated Using Explant and Enzymatic Methods You may also like Mesenchymal stem cell-derived extracellular matrix (mECM): a bioactive and versatile scaffold for musculoskeletal tissue.
      ]. Two studies have reported xeno/serum-free isolation of MSCs from adipose tissue. Sidhu et al. [
      • Sidhu H.
      • Talavera-Adame D.
      • Newman N
      Characterization of primary and immortalized human adipose stem cells cultured in a novel serum-free xeno-free media.
      ] used a self-made medium in combination with human fibronectin as surface coating. In the study by Hoang et al. [
      • Hoang V.T.
      • et al.
      Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.
      ], only the use of StemMACS XF in combination with CELLstart resulted in isolated cells showing MSC-specific CD marker expression, trilineage differentiation potential and self-renewal. Unfortunately, both reports lack information regarding isolated cell numbers or comparison with serum-containing control.
      To support the ongoing development of manufacturing processes based on well-defined or completely defined media for safe and high-quality MSCs, a surface coating based on human/animal component-free and chemically synthesized components that supports efficient isolation and expansion would be optimal. This prompted the authors to test the chemically defined myMATRIX MSC for the isolation of MSCs from bone marrow aspirates. Unfortunately, myMATRIX MSC provides only suboptimal support in BM-MSC isolation (see supplementary Figure 1). Hence, the authors hypothesized that a different microenvironment (i.e., a different coating composition with optimized attachment sites for the process of MSC isolation) would be necessary for successful isolation. Therefore, the authors developed isoMATRIX (denovoMATRIX GmbH), a chemically defined biomatrix specifically designed for the isolation of MSCs. Analogous to the authors’ previous strategy, isoMATRIX is composed of dextran sulfate and an ECM protein-derived peptide conjugate. Here the authors tested whether isoMATRIX supports the isolation of MSCs in xeno/serum-free conditions. The authors successfully isolated MSCs from bone marrow aspirates (0.2 MSCs per MNC) and observed an increase in isolation efficiency in comparison with isolation with serum-containing medium on uncoated culture vessels. Likewise, in combination with xeno/serum-free and chemically defined medium, the use of isoMATRIX was advantageous for the isolation of MSCs from subcutaneous fat. The isolated MSCs showed characteristic phenotype, high viability, high proliferation over five passages, strong differentiation potential and immunomodulatory capacities.

      Methods

      Isolation and culture of MSCs from bone marrow aspirates

      MSCs were isolated from healthy donors (n = 10) after obtaining informed written consent and according to a local research ethics board–approved protocol (ethical approval nos. EK221102004 and EK47022007). Briefly, human bone marrow aspirates were diluted in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA) at a ratio of 1:5. A 25-mL aliquot was layered over a 1.073-g·mL–1 Ficoll-Paque PLUS solution (GE Healthcare, Chicago, IL, USA) and centrifuged at 2000 × g for 25 min at room temperature. The mononuclear fraction was recovered, washed with PBS, and seeded as passage zero in either DMEM, low glucose, GlutaMAX supplement, pyruvate (Thermo Fisher Scientific) + 10% fetal calf serum (FCS) (Sigma-Aldrich, St Louis, MO, USA) on plastic or PRIME-XV MSC Expansion XSFM medium (FUJIFILM Irvine Scientific, Santa Ana, CA, USA) on isoMATRIX. Cells were cultured at 37°C and 5% CO2 in a humidified atmosphere and expanded further in the appropriate conditions. On day 4 after isolation, the cell number was determined by manually counting 10 × 13 tile images (×4) using Fiji software [
      • Schindelin J.
      • et al.
      Fiji: An open-source platform for biological-image analysis.
      ]. Cells were harvested using TrypLE Express Enzyme (Thermo Fisher Scientific) and subcultured in the same conditions. Viability, cell number and cell size were assessed using Trypan Blue dye exclusion with EVE cell counter (NanoEntek, Seoul, South Korea). A full medium change was performed twice a week.

      Flow cytometry

      For characterization of the MSC-specific surface phenotype, cells were incubated with fluorophore-coupled antibodies against CD11b, CD14, CD34, CD44, CD45, CD73, CD90, CD105, CD146 and CD166 or the corresponding isotype controls in PBS. For each sample, 30 000–50 000 events were acquired using BD FACSCanto fluorescence-activated cell sorting (BD Biosciences, Heidelberg, Germany). Histogram overlay subtraction analysis was performed using FlowJo software (BD Biosciences) to calculate the percentage of marker-positive cells.

      In vitro multi-lineage differentiation assays

      Cells were seeded in uncoated 24-well plates (serum-containing condition) or in plates coated with myMATRIX MSC (xeno/serum-free condition) at a density of 10 000 cells·cm–2 19. At 90% confluence, media were replaced by either complete adipogenesis medium (StemPro Adipogenesis Differentiation Basal Medium + StemPro Adipogenesis Supplement; Thermo Fisher Scientific) or osteogenesis medium (StemPro Osteocyte/Chondrocyte Differentiation Basal Medium + StemPro Osteogenesis Supplement; Thermo Fisher Scientific). Medium exchange was performed twice a week. For chondrogenic differentiation, micro-mass cultures of 1.6 × 107 cells were prepared and seeded as 5-µL droplets (80 000 cells per droplet) in the center of each well. After 2 h of cultivation, complete chondrogenesis medium (StemPro Osteocyte/Chondrocyte Differentiation Basal Medium + StemPro Chondrogenesis Supplement; Thermo Fisher Scientific) was added and exchanged every 2–3 days. Adipogenic differentiation was stopped after 1–2 weeks, osteogenic differentiation after 4–5 weeks and chondrogenic differentiation after 3 weeks. All samples were rinsed once with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The extent of differentiation was determined microscopically by the appearance of Oil Red O-stained lipid vacuoles in adipocytes, Alizarin Red-stained calcium deposits produced by osteocytes or Alcian Blue-stained proteoglycans synthesized by chondrocytes. Images were taken using a Lionheart FX microscope with Gen5 3.03 software (BioTek Instruments, Winooski, VT, USA).

      Colony-forming unit fibroblast assay

      A total of 130 cells (passages zero to one) were seeded in a T75 flask (TPP Techno Plastic Products AG, Trasadingen, Switzerland) in the appropriate condition and cultured for 14 days. The corresponding medium (DMEM + 10% FCS or PRIME-XV MSC Expansion XSFM medium) was changed after 7 days of culture. After 14 days, the colonies were washed once with PBS and stained with 0.05% Crystal Violet and counted.

      Lymphocyte stimulation assay

      To test the immunomodulatory potential of isolated MSCs, a lymphocyte stimulation and subsequent thymidine incorporation assay was performed as described previously [
      • Von Dalowski F.
      • et al.
      Mesenchymal Stromal Cells for Treatment of Acute Steroid-Refractory Graft Versus Host Disease: Clinical Responses and Long-Term Outcome.
      ]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteer donors after obtaining written consent (ethical approval no. EK206082008). The mononuclear cell fraction was isolated by Ficoll density gradient centrifugation, recovered, washed twice with PBS and resuspended in Roswell Park Memorial Institute 1640 supplemented with 10% FCS. Lymphocyte stimulation was performed by mixing 1 × 105 PBMCs, CD3/CD28 Dynabeads (Life Technologies, Carlsbad, CA, USA) and 5 × 103 MSCs irradiated at 30 Gy using a Gammacell 3000 Elan device (Best Theratronics Ltd, Ottawa, Canada). After 5 days of incubation at 37°C and 5% CO2 in a water-jacketed incubator, 1 µCi [3H]-thymidine (Hartmann Analytic, Braunschweig, Germany) was added to the culture. After an additional 18 h of incubation, cells were harvested and [3H]-thymidine incorporation was determined with a 1450 MicroBeta TriLux (PerkinElmer, Waltham, MA, USA), converting degree of radioactivity to counts per min.

      Isolation of adipose tissue-derived MSCs

      Full-thickness skin samples were obtained with informed consent of the donor according to the conditions listed in a valid ethics approval issued by the competent authority in the country of origin. Subcutaneous adipose tissue was minced and digested with collagenase I (Worthington Biochemical Corporation, Lakewood, NJ, USA) in PBS 2 mg/mL at 37 °C for 90 min. The digest was diluted 1:1 with CnT-PR-MSC-XF-HC (CELLnTEC, Bern, Switzerland), a chemically defined animal and human component-free culture medium, and the mix was permitted to stand undisturbed to allow for phase separation. The lower aqueous phase was sequentially strained through 100-µm and then 70-µm sieves. The resulting cell suspension was centrifuged, and the cell pellet was resuspended in one volume of CnT-PR-MSC-XF-HC before adding three volumes of 1X Red Blood Cell Lysis Buffer (BioLegend, San Diego, CA, USA). After 10 min of incubation at room temperature, another 10 volumes of CnT-PR-MSC-XF-HC were added and the cell suspension was centrifuged. The cell pellet was resuspended in CnT-PR-MSC-XF-HC and cells were counted. A total of 500 000 cells per T25 cell culture flask were seeded in CnT-PR-MSC-XF-HC, CnT-PR-MSC-XF (CELLnTEC) or DMEM, low glucose, GlutaMAX supplement, pyruvate + 10% FCS in either uncoated or isoMATRIX-coated flasks. Medium was changed the next day and then every 2–3 days. On day 6, the cells were detached using CnT-Accutase-100 (CELLnTEC) and counted.

      Illustrations and statistical analysis

      Illustrations were created with BioRender.com. Statistical analyses were performed using Prism 7.05 (GraphPad Software, San Diego, CA, USA). Outliers were evaluated using the ROUT method (Q = 1%). The significance of isolated cell numbers was assessed using the Mann–Whitney test. A two-tailed unpaired Student's t-test was applied for the evaluation of average cell size and number of colonies in colony-forming unit fibroblast assay. Differences were regarded as significant if the calculated P values were ≤ 0.05.

      Results

      Xeno/serum-free isolation of BM-MSCs with isoMATRIX results in a 4-fold increase in isolated cell number and significant decrease in cell size

      The authors previously developed myMATRIX MSC, a surface coating optimized for MSC expansion, which showed suboptimal support during MSC isolation from bone marrow aspirates (see supplementary Figure 1) [
      • Thamm K.
      • et al.
      A Novel Synthetic, Xeno-Free Biomimetic Surface for Serum-Free Expansion of Human Mesenchymal Stromal Cells.
      ]. To identify a new biomatrix that can aid MSC isolation, the authors selected 12 candidates—previously identified from the authors’ in-house screening tool (screenMATRIX; denovoMATRIX GmbH)—that supported efficient MSC expansion. In preliminary isolation tests, the use of a biomatrix composed of dextran sulfate and a fibronectin-derived peptide conjugate resulted in the typical fibroblast-like, spindle-shaped MSC morphology and highest isolated cell number (data not shown). Hence, this biomatrix was chosen for further development and tested for its ability to support the isolation of MSCs using xeno/serum-free and chemically defined medium. To isolate human BM-MSCs, MNCs were seeded in duplicate in uncoated six-well plates using serum-containing medium (tissue culture plastic [TCP] control) or in well plates coated with the new biomatrix, isoMATRIX, in combination with a xeno/serum-free medium. After 24 h of incubation, non-adherent cells were washed off and fresh medium was added. On day 4 post-isolation, the number of attached cells was manually counted in 10 × 13 tile images. An increase of approximately 30% in isolated cell numbers was detected when using isoMATRIX compared with the control condition (Figure 1A). This increase led to a significant 4-fold increment in the number of isolated MSCs on isoMATRIX compared with TCP control 11–14 days post-isolation (average ± standard deviation, 131.737 ± 184.801 on TCP, 543.889 ± 474.790 on isoMATRIX) (Figure 1B). In addition, cells showed high viability (Figure 1C) and the typical spindle-shaped, fibroblast-like morphology (Figure 1D) after isolation with isoMATRIX. Importantly, the authors were not able to isolate any cells using uncoated or fibronectin-coated culture vessels.
      Fig 1
      Fig. 1Increased isolated cell numbers, high viability and decreased cell size of BM-MSCs isolated using isoMATRIX in xeno/serum-free conditions. MNCs were seeded in duplicate in uncoated (TCP) or isoMATRIX-coated six-well plates using serum-containing medium and xeno/serum-free medium, respectively. No cells were isolated from TCP or fibronectin plates in combination with xeno/serum-free medium. After 24 h, non-adherent cells were washed off and fresh medium was added. (A) On day 4 after isolation, the cell number was determined by manual counting of 10 × 13 tile images using Fiji software. After 11–14 days, cells were harvested and the (B) number, (C) viability, (D) morphology and (E) size of isolated MSCs were determined. Cell size distribution of BM-MSCs isolated in xeno/serum-free medium in combination with isoMATRIX and serum-containing medium without any coating was analyzed by (F,G) cell counter and (H,I) flow cytometry (n = 10). Scale bar = 200 µm. Mann–Whitney test and two-tailed unpaired Student's t-test, respectively, were used to determine significance of isolated cell number after (B) 11–14 days and (E) determination of average cell size. ***P < 0.005, ****P < 0.0001. FSC-A, forward scatter area; SSC-A, side scatter area. (Color version of figure is available online.)
      The authors and others have previously reported that BM-MSCs expanded in serum-free medium show a smaller cell size than MSCs expanded in serum-containing medium [
      • Thamm K.
      • et al.
      A Novel Synthetic, Xeno-Free Biomimetic Surface for Serum-Free Expansion of Human Mesenchymal Stromal Cells.
      ,
      • Chase L.G.
      • Lakshmipathy U.
      • Solchaga L.A.
      • Rao M.S.
      • Vemuri M.C
      A novel serum-free medium for the expansion of human mesenchymal stem cells.
      ]. Similarly, cells isolated using isoMATRIX in combination with xeno/serum-free medium were significantly smaller (16 ± 0.8 µm) compared with those isolated under serum-containing conditions (20 ± 0.8 µm) (Figure 1E). The high growth rates of isoMATRIX-derived MSCs support the concept that small cell size indicates high proliferative activity and low senescence [

      Kim, M. et al. A Small-Sized Population of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Shows High Stemness Properties and Therapeutic Benefit. Stem Cells International, vol. 2020, Article ID 5924983, 17 pages

      ,
      • Ng C.P.
      • et al.
      Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM.
      ]. Moreover, the isolation of cells using isoMATRIX with xeno/serum-free medium resulted in a more homogeneous cell population, with a cell size of 10–22 µm for most cells (96.4% of total counted cells) (Figure 1F) compared with 10–30 µm for control cells (96.7% of total counted cells) (Figure 1G). This increase in homogeneity of cells isolated using isoMATRIX was also reflected in flow cytometry-based size distribution (Figure 1H).
      In sum, the authors successfully isolated MSCs from bone marrow aspirates in xeno/serum-free conditions using isoMATRIX. In addition, the authors observed a strong increase in isolation efficiency in comparison with isolation with serum-containing medium on uncoated culture vessels.

      BM-MSCs isolated under xeno/serum-free conditions maintain characteristic expression profiles of cell surface antigens

      The expression of specific cell surface antigens is an essential criterion for the characterization of human BM-MSCs. According to the International Society for Cell & Gene Therapy (ISCT), BM-MSCs isolated and expanded in serum-containing media on a plastic surface should express CD73, CD105 and CD90 in at least 95% of the cell population. By contrast, they must not express the hematopoietic markers CD45, CD34, CD11b or CD14, CD79α or CD19 and HLA-DR (<2% positive) [
      • Dominici M.
      • et al.
      Minimal criteria for defining multipotent mesenchymal stromal cells . The International Society for Cellular Therapy position statement.
      ]. In addition, other cell surface markers, including CD44, CD146, Stro-1 and CD271, have been used to enrich a human MSC population with trilineage differentiation and colony-forming abilities [
      • Lv Feng-Juan
      • Tuan Rocky S.
      • Cheung Kenneth M.C.
      • Leung V.Y.L
      Consise Review: The Surface Markers and Identity of Human Mesenchymal Stem Cells.
      ].
      To test whether cells isolated in xeno/serum-free conditions on isoMATRIX show MSC-specific cell surface antigens, the authors examined the expression of 10 cell surface makers (CD73, CD105, CD90, CD44, CD146, CD166, CD11b, CD14, CD45 and CD34) by flow cytometry using cells of passages two and three (Figure 2). Almost all cells (99 ± 1.6%) isolated in xeno/serum-free medium using isoMATRIX were positive for CD73, CD105, CD90 and CD44. In addition, they showed high levels of CD146 (92 ± 6%) and CD166 (78 ± 11%) (Figure 2A) and did not express CD34, CD11b, CD14 and CD45 (1.0 ± 0.8%, 0.8 ± 0.2%, 0.6 ± 0.3% and 0.2 ± 0.3%, respectively) (Figure 2B). As postulated by the ISCT, >95% of TCP-isolated cells expressed CD73, CD105 and CD90 (Figure 2C), and 98.9% also expressed CD44. These cells showed a similar expression level of CD146 (93.5 ± 6.4%) but a higher expression of CD166 (98.7 ± 0.9%) compared with cells isolated under xeno/serum-free conditions. In TCP-isolated cells, the expression of hematopoietic markers CD34, CD11b, CD14 and CD45 was higher compared with isoMATRIX-derived BM-MSCs (3.1 ± 1.3%, 2.5 ± 1.3%, 3.1 ± 3.8% and 1.4 ± 1.0%, respectively), often exceeding the 2% limit postulated by the ISCT (Figure 2D). Thus, BM-MSCs isolated under xeno/serum-free conditions with isoMATRIX fulfill the requirements of international guidelines, with better delineation from hematopoietic stem cells.
      Fig 2
      Fig. 2Expression profiles of cell surface antigens of BM-MSCs. Cells isolated and expanded to passages two to three in either (A,B) xeno/serum-free medium on isoMATRIX (n = 6) or (C,D) serum-containing medium on TCP (n = 5) were examined by flow cytometry for the expression of cell surface markers CD73, CD105, CD90, CD44, CD146, CD166, CD11b, CD14, CD45 and CD34. The average percentage of cells positive for the indicated marker and standard deviation are indicated. (Color version of figure is available online.)

      BM-MSCs isolated under xeno/serum-free conditions using isoMATRIX show significantly enhanced colony-forming efficiency and high trilineage differentiation potential and retain immunomodulatory capacity

      Next, the authors investigated whether cells isolated with isoMATRIX and XSFM medium exhibit self-renewal and immunomodulatory and trilineage differentiation potential—hallmarks of MSCs. First, the authors evaluated the clonogenic potential of BM-MSCs (passages zero to one) using a colony-forming unit fibroblast assay. A total of 130 cells were seeded in a T75 flask and cultured for 14 days in the corresponding condition, and the number of colonies was counted after Crystal Violet staining. As shown in Figure 3A–C, significantly more proliferative and adherent cells could be effectively isolated using isoMATRIX combined with xeno/serum-free medium (39 ± 7 colonies) compared with the serum-containing method (0.9 ± 1.5 colonies).
      Fig 3
      Fig. 3Enhanced colony-forming efficiency, high immunomodulatory capacity and trilineage differentiation potential of isoMATRIX-derived BM-MSCs. BM-MSCs isolated and expanded in either xeno/serum-free medium using isoMATRIX or serum-containing medium on TCP were used to perform (A–C) CFU-F assay, (D) lymphocyte stimulation assay and (E–G) assessment of trilineage differentiation potential. (A–C) A total of 130 cells (passages zero to one) were seeded in a T75 flask, cultured for 14 days in xeno/serum-free medium on isoMATRIX (duplicates shown) or serum-containing medium on TCP and stained with Crystal Violet for analysis. (D) For lymphocyte stimulation assay, cells (passages two to three) were irradiated and incubated with CD3/CD28-stimulated PBMCs. Bars represent the relative proliferative capacity of cells displaying [3H]-thymidine incorporation compared with control (PBMCs alone = 1.0). (E–G) For analysis of adipogenic, osteogenic and chondrogenic differentiation, cells were stained with Oil Red O for lipid droplets after 7 days, Alizarin Red for calcium phosphate deposits produced by osteocytes after 28 days and Alcian Blue for proteoglycans synthesized by chondrocytes after 21 days of induction. Images were taken using a Lionheart FX microscope (×4). Representative images are shown (n = 4). Scale bar = 200 µm. CFU-F, colony-forming unit fibroblast. (Color version of figure is available online.)
      To evaluate the immunomodulatory properties of BM-MSCs, the authors examined their impact on PBMC proliferation. BM-MSCs of four different donors were used to perform a lymphocyte stimulation assay [
      • Von Dalowski F.
      • et al.
      Mesenchymal Stromal Cells for Treatment of Acute Steroid-Refractory Graft Versus Host Disease: Clinical Responses and Long-Term Outcome.
      ]. Similar to TCP-isolated cells, isoMATRIX-derived BM-MSCs were able to reduce the relative proliferative capacity to 0.3–0.5 (Figure 3D). Thus, MSCs obtained from bone marrow aspirates under xeno/serum-free isolation conditions maintain immunomodulatory capacities comparable to cells isolated under serum-containing conditions. isoMATRIX-derived BM-MSCs were also differentiated toward adipocytes, osteocytes and chondrocytes to assess their trilineage differentiation potential. The adipogenic differentiation was visualized by staining cytoplasmic lipid droplets with Oil Red O after 7 days (Figure 3E). Osteogenic and chondrogenic differentiation was observed by Alizarin Red staining of calcium phosphate deposits after 28 days and Alcian Blue staining of proteoglycans after 21 days, respectively (Figure 3F,G). All tested donors showed a high ability to differentiate into the three mesoderm-specific lineages. In sum, the authors’ results demonstrate that isolation of MSCs from bone marrow aspirates with isoMATRIX under xeno/serum-free conditions extracts a cell population with high proliferation and differentiation potential as well as strong immunomodulatory capacities.

      isoMATRIX improves the efficiency of MSC isolation from adipose tissue in xeno-free and chemically defined medium

      MSCs isolated from different tissue sources share a common phenotype (Figures 1D, 4A) and differentiation potential as well as the expression of many cell surface markers but differ in gene expression patterns, proliferation rate and therapeutic efficacy [
      • Kern S.
      • Eichler H.
      • Stoeve J.
      • Klüter H.
      • Bieback K.
      Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood, or Adipose Tissue.
      ,
      • Wagner W.
      • et al.
      Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.
      ,
      • Ribeiro A.
      • et al.
      Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells.
      ]. To study the ability of isoMATRIX to support the isolation of MSCs from other tissue sources, the authors performed an experiment using subcutaneous adipose tissue from a single donor. In addition, the authors wanted to test isoMATRIX in combination with different media and hence used a different xeno-free medium as well as a chemically defined medium. Six days after isolation, the number of cells was markedly increased in both media on isoMATRIX compared with the uncoated control (TCP) and the serum-containing condition (Figure 4B). The highest fold change (2.5-fold) in the number of isolated adipose tissue-derived MSCs was observed using the chemically defined medium in combination with isoMATRIX (CnT-PR-MSC-XF-HC + isoMATRIX, 8.15 ± 1.13 × 105 cells, TCP, 3.31 ± 0.3 × 105 cells). A significant increase in isolation efficiency was also seen under xeno-free conditions, with a 1.7-fold increase in isolated cell number (CnT-PR-MSC-XF + isoMATRIX, 1.26 ± 0.06 × 106 cells, TCP, 7.23 ± 0.08 × 105 cells). These results indicate that isoMATRIX also enhances the isolation of MSCs from adipose tissue and underline its compatibility with diverse media, ranging from xeno/serum-free to chemically defined.
      Fig 4
      Fig. 4Isolation of MSCs from subcutaneous adipose tissue. Cells were isolated from the stromal vascular fraction of subcutaneous adipose tissue samples after enzymatic tissue digestion by seeding 20 000 cells/cm2 on either isoMATRIX or TCP in combination with xeno-free (CnT-PR-MSC-XF), chemically defined (CnT-PR-MSC-XF-HC) or serum-containing (DMEM + FCS) medium. (A) Morphology and (B) cell number were analyzed on day 6 post-isolation (n = 1). Scale bar = 300 µm. (Color version of figure is available online.)

      BM-MSCs isolated under xeno/serum-free conditions maintain high proliferation

      Using isoMATRIX in combination with XSFM medium for bone marrow isolation resulted in cells with MSC-specific characteristics (Figures 2, 3) and a high proliferative potential in passage zero compared with cells isolated under serum-containing conditions (Figure 1B). To investigate whether these cells maintain their proliferative potential during subsequent cultivation, the authors expanded them for an additional five passages. Moreover, the authors tested whether isoMATRIX could provide a proliferative advantage for MSCs isolated in serum-containing medium.
      Six different donors were isolated in either serum-containing medium or XSFM on TCP or isoMATRIX. After isolation with isoMATRIX, cells were either expanded in their isolation conditions or transferred to myMATRIX MSC, a specific cell culture coating supporting MSC proliferation [
      • Thamm K.
      • et al.
      A Novel Synthetic, Xeno-Free Biomimetic Surface for Serum-Free Expansion of Human Mesenchymal Stromal Cells.
      ]. BM-MSCs isolated in serum-containing medium on TCP were expanded in their isolation conditions or transferred to xeno/serum-free conditions (Figure 5A). Cell number was counted manually at day 4, and cells were harvested at 80–90% confluence to determine final cell number and fold expansion. In passage zero, the authors observed an average fold expansion of 3246 ± 2135 for XSFM on isoMATRIX, 904 ± 665 for DMEM + FCS on isoMATRIX and 363 ± 261 for DMEM + FCS on TCP (Figure 5B). Thus, isolating cells in xeno/serum-free conditions using isoMATRIX resulted in 10 times more cells in passage zero compared with serum-containing conditions on TCP. Using isoMATRIX in combination with serum-containing medium for isolation of BM-MSCs doubled the cell yield at passage zero compared with isolation on TCP. Moreover, BM-MSCs isolated in xeno/serum-free conditions on isoMATRIX reached confluence on average 5–6 days earlier compared with serum-containing isolation conditions (XSFM + isoMATRIX, 11 ± 1.7 days, serum-containing medium + isoMATRIX, 16 ± 2.1 days, serum-containing medium + TCP, 17 ± 1.5 days).
      Fig 5
      Fig. 5High proliferation of BM-MSCs isolated and expanded in xeno/serum-free conditions. BM-MSCs were isolated using serum-containing (DMEM) or xeno/serum-free (XSFM) medium in combination with TCP or isoMATRIX. No cells could be isolated in XSFM on TCP. Isolated cells were further expanded in their isolation conditions or transferred to xeno/serum-free medium in combination with isoMATRIX or myMATRIX MSC. (A) Schematic representation of the experiment. Fold expansion of cell numbers was determined for (B) passage zero and (C) passages one through five (donor three). Linear regression model was used to indicate the fold expansion trend line. Total cell number was calculated for each condition for the (D) first 3–4 weeks after isolation and the (E) entire culture period (donor five). Data for all donors can be found in supplementary Figures 2–4. (Color version of figure is available online.)
      Cell expansion over five consecutive passages demonstrated the well-known donor variability. Thus, donors showed diverse growth profiles and varying proliferation rates in the different culture conditions. Figure 5C shows a representative profile of fold expansion during the culture period of five passages. In general, cell expansion in serum-containing medium resulted in overall low growth, with an average fold expansion of 3.4 ± 4.2 throughout the five passages (see supplementary Figure 2). Expansion on isoMATRIX or myMATRIX MSC only slightly improved the growth rates of three donors when using serum-containing medium. By contrast, expansion of BM-MSCs in xeno/serum-free conditions showed an average fold expansion of 10.7 ± 6.3 during the culture period. Most of the donors showed a reduction in proliferation potential with time, with varying degrees of deterioration, irrespective of the culture condition (see supplementary Figure 2). Interestingly, the transfer of BM-MSCs isolated in serum-containing medium on TCP into xeno/serum-free conditions for expansion often showed a peak in fold expansion in passage one with a steep decline during subsequent passages (Figure 5C). The authors speculate that the cells might carry over FCS, which, in combination with the xeno/serum-free environment, leads to a significant increase in proliferation in the first passage after the transfer.
      To illustrate the total number of cells that could have been produced (per passage) if all cells of the previous passage had been used as input material, the authors multiplied the number of harvested cells at passage zero by the values of the fold expansion per passage. Using isoMATRIX for isolation and myMATRIX MSC for expansion in xeno/serum-free medium led to the highest cell yield for five of the six tested donors 3–4 weeks after isolation (Figure 5D; also see supplementary Figure 3). This translated to the highest total cell number after five passages for three donors when cultured in these conditions (Figure 5E; also see supplementary Figure 3). For the other three donors, isolation in serum-containing medium on TCP and expansion in XSFM medium on myMATRIX MSC produced the highest number of cells at passage five.
      In sum, BM-MSCs isolated and expanded in xeno/serum-free conditions with isoMATRIX and myMATRIX MSC maintain high proliferation during subsequent passages, resulting in fast and efficient cell production. In addition, isoMATRIX supports the isolation of MSCs in serum-containing media.

      Discussion

      MSC-based therapies are used to treat a variety of diseases, including Alzheimer disease [
      • Nakano M.
      • et al.
      Bone marrow-derived mesenchymal stem cells improve cognitive impairment in an Alzheimer's disease model by increasing the expression of microRNA-146a in hippocampus.
      ,
      • Zhang L.
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      Immunomodulatory role of mesenchymal stem cells in Alzheimer's disease.
      ,
      • Guo M.
      • Yin Z.
      • Chen F.
      • Lei P.
      Mesenchymal stem cell-derived exosome: A promising alternative in the therapy of Alzheimer's disease.
      ], bone and cartilage diseases [
      • Kangari P.
      • Talaei-Khozani T.
      • Razeghian-Jahromi I.
      • Razmkhah M.
      Mesenchymal stem cells: amazing remedies for bone and cartilage defects.
      ,
      • Undale A.H.
      • Westendorf J.J.
      • Yaszemski M.J.
      • Khosla S.
      Mesenchymal stem cells for bone repair and metabolic bone diseases.
      ,
      • Zhang R.
      • Ma J.
      • Han J.
      • Zhang W.
      • Ma J
      Mesenchymal stem cell related therapies for cartilage lesions and osteoarthritis.
      ], autoimmune diseases [
      • Munir H.
      • McGettrick H.M.
      Mesenchymal Stem Cell Therapy for Autoimmune Disease: Risks and Rewards.
      ,
      • Rad F.
      • Ghorbani M.
      • Mohammadi Roushandeh A.
      • Habibi Roudkenar M.
      Mesenchymal stem cell-based therapy for autoimmune diseases: emerging roles of extracellular vesicles.
      ], diabetes [
      • Wang L.
      • et al.
      Mesenchymal stem cells ameliorate β cell dysfunction of human type 2 diabetic islets by reversing β cell dedifferentiation.
      ,
      • Pixley J.S.
      Mesenchymal stem cells to treat type 1 diabetes.
      ], graft-versus-host disease [
      • Amorin B.
      • et al.
      Mesenchymal stem cell therapy and acute graft-versus-host disease: a review.
      ,
      • Elgaz S.
      • Kuçi Z.
      • Kuçi S.
      • Bönig H.
      • Bader P.
      Clinical Use of Mesenchymal Stromal Cells in the Treatment of Acute Graft-versus-Host Disease.
      ] and multiple sclerosis [
      • Dulamea A.
      Mesenchymal stem cells in multiple sclerosis - translation to clinical trials.
      ,
      • Scolding N.J.
      • Pasquini M.
      • Reingold S.C.
      • Cohen J.A.
      Cell-based therapeutic strategies for multiple sclerosis.
      ]. MSCs offer many advantages for clinical applications, such as easy accessibility, straightforward isolation and expansion procedures, high proliferative capacities, safety in autologous and allogeneic therapies and preservation of potency after storage. However, because of the lack of standardized procedures for harvest, cultivation and functional characterization as well as differences between donors and tissues of origin, it is still challenging to manufacture an MSC-based therapeutic product with consistent high yield and quality. Hence, setting up a reliable, consistent and scalable system is critical for producing safe and potent MSCs on a broader scale despite the complex manufacturing process. Avoiding the use of serum-containing medium during both cell isolation and expansion is not only beneficial for product safety but also helps to reduce lot-to-lot inconsistencies and enables commercialization of MSC-based cell therapies on a large scale.
      The application of xeno/serum-free culture medium is a first step toward highly defined and thus more consistent manufacturing processes. Unfortunately, it can lead to reduced cell attachment and proliferation due to the lack of adhesion molecules, growth factors and/or nutrients. To support the use of xeno/serum-free media without the need for human blood-derived supplements or animal-derived protein coatings, the authors recently developed a chemically defined biomatrix (myMATRIX MSC) that promotes cell adhesion and supports the long-term expansion of MSCs with robust growth and high viability while maintaining their differentiation and immunomodulatory capacity, characteristic cell morphology and expression of key stemness markers [
      • Thamm K.
      • et al.
      A Novel Synthetic, Xeno-Free Biomimetic Surface for Serum-Free Expansion of Human Mesenchymal Stromal Cells.
      ]. However, the use of myMATRIX MSC for the isolation of cells from bone marrow aspirates resulted in suboptimal cell morphology and low isolation efficiency. Based on these observations, the authors hypothesized that a different biomatrix was required during the isolation phase to better support initial attachment of cells transitioning from the in vivo to in vitro microenvironment.
      To the authors’ knowledge, no data have been published on the use of a chemically defined surface coating for the isolation of MSCs. The authors therefore developed an isolation-optimized, ECM-mimetic biomatrix called isoMATRIX, which is composed of dextran sulfate and an ECM protein-derived peptide conjugate. Using isoMATRIX in combination with xeno/serum-free PRIME-XV MSC Expansion XSFM medium for the isolation of MSCs from bone marrow aspirates resulted in an enhanced isolation efficiency of approximately 30% on day 4, which led to a 4- to 10-fold increase in the number of harvested cells on day 11 to day 14 (passage zero) compared with serum-based isolation on TCP. To a lesser extent, isoMATRIX also increased the number of harvested cells in passage zero (2.5-fold) when serum-containing medium was used. Importantly, the authors were not able to isolate any cells using uncoated or fibronectin-coated culture vessels. The authors’ experiments showed an average isolation efficiency (number of MSCs per MNC) in xeno/serum-free medium of 0.2 at passage zero, which was doubled compared with the recently reported value of approximately 0.1 by Hoang et al. [
      • Hoang V.T.
      • et al.
      Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.
      ].
      Since MSCs can be isolated from various body tissues, the authors also tested isoMATRIX using subcutaneous adipose tissue in combination with another xeno-free medium as well as a chemically defined medium. Irrespective of the medium, isoMATRIX markedly increased cell yield in both conditions compared with uncoated controls, with a fold increase in isolated cell numbers ranging from 1.7 to 2.5. These results indicate that isoMATRIX can strongly enhance the isolation efficiency of MSCs using diverse media and different tissue sources. Furthermore, they illustrate that process-specific biomatrices may be beneficial for MSC manufacturing pipelines. It is also likely that further optimization of the surface coating (and medium) toward tissue-specific requirements would result in even higher cell yields after isolation of MSCs from adipose tissue or the like, resulting in maximized process efficiency.
      The population of BM-MSCs isolated with isoMATRIX was more homogeneous and significantly smaller than serum-isolated controls but displayed the typical spindle-shaped, fibroblast-like morphology. The size of MSCs is often reported to correlate with their self-renewing properties and/or differentiation potential, with larger cells showing a lower proliferation and differentiation capacity and increased senescence [
      • Bhartiya D.
      • et al.
      Very Small Embryonic-Like Stem Cells with Maximum Regenerative Potential Get Discarded During Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy.
      ,
      • Liu J.
      • Ding Y.
      • Liu Z.
      • Liang X.
      Senescence in Mesenchymal Stem Cells: Functional Alterations, Molecular Mechanisms, and Rejuvenation Strategies.
      ,
      • Lee W.C.
      • et al.
      Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency.
      ]. Interestingly, Yin et al. [
      • Yin L.
      • et al.
      Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential in vitro.
      ] showed that continuous selection for a homogeneous and smaller population of MSCs results in faster proliferation and higher chondrogenic potential compared with conventional expansion procedures. In the authors’ present study, the smaller and more homogeneous cell population isolated with isoMATRIX also showed a higher clonogenic and trilineage differentiation potential as well as a strong immunomodulatory capacity. In addition, isoMATRIX-derived MSCs expressed the characteristic profile of MSC-specific cell surface antigens. Similar to serum-isolated control cells, >95% expressed CD73, CD105 and CD90, as postulated by the ISCT. They also showed no difference in the expression of CD44 and CD146. By contrast, the authors found elevated expression levels of CD34, CD11b and CD14 in BM-MSCs isolated using TCP and serum-containing medium that exceeded the limit of 2%, as defined by the ISCT. The expression of CD166 was reduced by approximately 20% in BM-MSCs isolated under xeno/serum-free conditions using isoMATRIX compared with the serum-containing condition. CD166 is a transmembrane protein of unknown function in MSC biology, and although significant variations have been observed in CD166 expression, it has been identified as a possible human MSC gene expression or surface marker [
      • Brinkhof B.
      • Zhang B.
      • Cui Z.
      • Ye H.
      • Wang H.
      ALCAM (CD166) as a gene expression marker for human mesenchymal stromal cell characterisation.
      ,
      • De Oliveira GLV
      • De Lima KWA
      • Colombini AM
      • et al.
      Bone Marrow Mesenchymal Stromal Cells Isolated from Multiple Sclerosis Patients have Distinct Gene Expression Profile and Decreased Suppressive Function Compared with Healthy Counterparts.
      ,
      • S J.
      • N H.
      • A C.
      • F D
      The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells.
      ,
      • Halfon S.
      • Abramov N.
      • Grinblat B.
      & Ginis, I. Markers Distinguishing Mesenchymal Stem Cells from Fibroblasts Are Downregulated with Passaging.
      ]. Based on the characterization assays that were performed, the authors could not specifically link the reduction in CD166 surface expression to any of the MSC properties investigated.
      In sum, the authors’ results suggest that xeno/serum-free isolation conditions might extract a more uniform cell population and/or that the xeno/serum-free microenvironment could reduce the cellular and metabolic heterogeneity that accumulates during in vitro expansion [
      • Liu Y.
      • Muñoz N.
      • Bunnell B.A.
      • Logan T.M.
      • Ma T.
      Density-Dependent Metabolic Heterogeneity in Human Mesenchymal Stem Cells.
      ,
      • Wilson A.
      • Hodgson-Garms M.
      • Frith J.E.
      • Genever P.
      Multiplicity of mesenchymal stromal cells: Finding the right route to therapy.
      ]. To confirm these initial indications, further comparative investigations, including cytokine secretion, messenger RNA expression and metabolite analysis, would be needed.
      Yuan et al. [
      • Yuan X.
      • Logan T.M.
      • Ma T.
      Metabolism in human mesenchymal stromal cells: A missing link between HMSC biomanufacturing and therapy?.
      ] discussed that the transfer of MSCs from their native microenvironment to non-physiological culture conditions with nutrient-rich medium, lack of cell–cell and cell–ECM connections and different geometric and mechanical properties results in a change in their energy metabolism from primarily glycolytic with active autophagy to a higher dependence on oxidative phosphorylation with reduced autophagy. This leads to increased senescence and reduced clinical potency of the cells as well as higher cellular and metabolic heterogeneity. Thus, a more accurate recreation of the natural microenvironment of human MSCs in vitro might lead to a more homogeneous and potent cell population. The superior isolation efficiency of isoMATRIX compared with TCP highlights the benefit of a carefully tailored cell microenvironment.
      The enhanced isolation efficiency is accompanied by high proliferation rates in subsequent passages. After isolation with isoMATRIX, the authors either cultured the cells in their isolation conditions or transferred them to myMATRIX MSC for an additional five passages. Cells isolated using serum-containing medium with TCP were additionally expanded in xeno/serum-free conditions in combination with isoMATRIX or myMATRIX MSC. Although cells proliferated during subsequent culture in XSFM medium on isoMATRIX, a switch to myMATRIX MSC for cell expansion appeared to be beneficial for all donors tested and resulted in highest total cell number for half of the donors. The authors also observed enhanced proliferation after switching from serum-containing medium with TCP to xeno/serum-free conditions, in which the use of myMATRIX MSC resulted in similar or higher cell growth compared with isoMATRIX. Interestingly, 50% of the tested donors showed highest total cell number when isolated in serum-containing medium on TCP and expanded in XSFM medium on myMATRIX MSC, but at clinically relevant times (within 3 weeks), isoMATRIX-isolated cells showed superior fold expansion. However, BM-MSCs isolated in serum-containing conditions might have harbored a high proliferative potential that they retained until transfer into an expansion-supporting microenvironment, or the switch in cultivation conditions could have led to a shift in cell identity. To be able to exclude the latter and confirm cellular integrity, all cells would need to be tested for MSC-specific characteristics after expansion. In sum, these results illustrate the sensitivity of the cells toward their microenvironment, the importance of the choice of medium in achieving appropriate expansion rates and the strong impact of the provided artificial surface on different cell culture procedures (isolation or expansion).
      Depending on the tissue of origin, there is a substantial difference in the number of MSCs that can be isolated; however, in any case, they are a rare population of progenitors in adult tissues. Consequently, the quantity of cells obtained after isolation is often insufficient for a clinical dose of approximately 106–109 cells [
      • Kabat M.
      • Bobkov I.
      • Kumar S.
      • Grumet M.
      Trends in mesenchymal stem cell clinical trials 2004-2018: Is efficacy optimal in a narrow dose range?.
      ,
      • Barekzai J.
      • Petry F.
      • Zitzmann J.
      • Czermak P.
      • Salzig D.
      Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products.
      ]. This problem is potentiated by the decrease in growth kinetics, differentiation ability and potency of MSCs during extensive expansion in vitro. Hence, efficient MSC isolation is the premise for the optimized expansion procedures required to produce high-quality cells in clinically relevant numbers. To illustrate the impact of enhanced isolation efficiency on the manufacturing process of an MSC-based therapeutic product, the authors applied the fold expansion data of the passaging experiment with donor 1 using myMATRIX and XSFM medium, which exemplified a highly efficient expansion process (see supplementary Figure 5A). The authors fitted an exponential growth curve and employed the resulting formula to model the clinically relevant manufacturing process of a therapeutic dose of 108 (Figure 6; also see supplementary Figure 5B).
      Fig 6
      Fig. 6Modeling the expansion process for an MSC-based therapeutic product. The authors assume a therapeutic dose of 108. The manufacturing process starts with an isolated cell population that covers a T75 flask (clinical application scale), which is propagated by stepwise surface enlargement until administration of cells to a patient (seeding density, 5000 cells/cm2, density at confluence, 40 000 cells/cm2 throughout the process). For autologous therapies, a 30% increase in starting material would shorten manufacturing time and allow the production of 50% of the cell number required for a therapeutic dose at passage two. The impact of a 30% increase in starting material on the manufacturing process of human MSCs for allogeneic therapies allows for serving an additional patient at passage two (indicated in blue). The expansion process model is inferred from the growth curve of donor one isolated with isoMATRIX and expanded on myMATRIX MSC in xeno/serum-free medium. The growth curve data were fitted to the exponential growth model Y = Y0e(kx) (non-linear fitting) with R2 = 0.9775. (Color version of figure is available online.)
      Assuming the isolation process would result in the number of cells sufficient for a T75 flask (Figure 6), the enhanced isolation efficiency provided by isoMATRIX in combination with a xeno/serum-free medium of, on average, 30% would result in an additional T25 flask covered in cells. Because of their extremely slow expansion after isolation, a comparison with cells isolated and expanded in serum-containing conditions did not appear feasible. Furthermore, a comparison with fibronectin-coated or uncoated vessels in combination with XSFM medium was not possible since no cells could be isolated under these conditions. The 30% improvement has several implications for the manufacturing process of human MSCs for autologous and allogeneic therapies. Autologous therapies are often restricted by the patient's low MSC frequency and/or poor quality due to age or disease state. Hence, increasing isolation efficiency accompanied by enhanced cell proliferation enables treatment of the patient in a shorter time period.
      In allogeneic stem cell therapy, cells from one donor are utilized for multiple patients and provided as an off-the-shelf product. The increase in isolated cell number provided by isoMATRIX at the beginning of the manufacturing process of such therapies can substantially increase the final cell yield, which is naturally limited by the lifetime of MSCs. In the authors’ model, an increase of 30% in starting material would result in sufficient cells to treat one additional patient after expanding the cells for two passages (seeding density, 5000 cells/cm2, density at confluence, 40 000 cells/cm2 throughout the process) (Figure 6). This impact would be potentiated in large-scale manufacturing, which would help reduce costs and expand patient access to MSC-based cell therapy. To support large-scale MSC expansion in bioreactor setups, the authors applied an approach similar to isoMATRIX/myMATRIX MSC to develop a ready-to-use microcarrier for three-dimensional culture called beadMATRIX (denovoMATRIX GmbH). Thus, the authors’ chemically defined biomatrix combined with xeno/serum-free or chemically defined media promotes enhanced isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

      Funding

      denovoMATRIX GmbH was supported by and received funding from the European Social Fund, European Regional Development Fund and EXIST Transfer of Research granted by the Federal Ministry for Economic Affairs and Energy.

      Declaration of Competing Interest

      KT, SS and RW are employees of denovoMATRIX GmbH. SB is an employee of CELLnTEC Advanced Cell Systems AG.

      Author Contributions

      Conception and design of the study: KT, RW and SS. Acquisition of data: KT, KM, RT and SB. Analysis and interpretation of data: KT, KM, RT and SB. Drafting or revising the manuscript: KT, RW, MW and SS. All authors have approved the final article.

      Appendix. Supplementary materials

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