Highlights
- •Flow-through device recovers more lymphocytes than density gradient centrifugation.
- •Cell recoveries were 85% CD3+, 89% CD19+ and 97% CD56+ with this new approach.
- •Microfluidic-isolated cells grew faster than gradient-isolated cells in culture.
- •Overall, the new method gives 2X higher cumulative cell yield after 7-day culture.
- •Devices can process 200 mL of sample in less than 1 h, with no pumping mechanism needed.
Abstract
Background
The isolation of lymphocytes – and removal of platelets (PLTs) and red blood cells
(RBCs) – from an initial blood sample prior to culture is a key enabling step for
effective manufacture of cellular therapies. Unfortunately, currently available methods
suffer from various drawbacks, including low cell recovery, need for complex equipment,
potential loss of sterility and/or high materials/labor cost.
Methods
A newly developed system for selectively concentrating leukocytes within precisely
designed, but readily fabricated, microchannels was compared with conventional density
gradient centrifugation with respect to: (i) ability to recover lymphocytes while
removing PLTs/RBCs and (ii) growth rate and overall cell yield once expanded in culture.
Results
In the optimal embodiment of the new microfluidic approach, recoveries of CD3+, CD19+
and CD56+ cells (85%, 89% and 97%, respectively) were significantly higher than for
paired samples processed via gradient-based separation (51%, 53% and 40%). Although
the removal of residual PLTs and RBCs was lower using the new approach, its enriched
T-cell fraction nevertheless grew at a significantly higher rate than the gradient-isolated
cells, with approximately twice the cumulative cell yield observed after 7 days of
culture.
Discussion
The standardization of each step of cellular therapy manufacturing would enable an
accelerated translation of research breakthroughs into widely available clinical treatments.
The high-throughput approach described in this study – requiring no ancillary pumping
mechanism nor expensive disposables to operate – may be a viable candidate to standardize
and streamline the initial isolation of lymphocytes for culture while also potentially
shortening the time required for their expansion into a therapeutic dose.
Graphical Abstract
Graphical Abstract
Key Words
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to CytotherapyAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- The Principles of Engineering Immune Cells to Treat Cancer.Cell. 2017; 168: 724-740
- Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.N Engl J Med. 2017; 377: 2531-2544
- Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.N Engl J Med. 2018; 378: 439-448
- T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.Blood. 2016; 128: 1688-1700
- Chimeric antigen receptor T cell therapy: 25 years in the making.Blood Rev. 2016; 30: 157-167
- Global Manufacturing of CAR T Cell Therapy.Mol Ther Methods Clin Dev. 2017; 4: 92-101
- Towards a commercial process for the manufacture of genetically modified T cells for therapy.Cancer Gene Ther. 2015; 22: 72-78
- Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells.Cytotherapy. 2016; 18: 893-901
- Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.Cytotherapy. 2017; 19: 433-439
- [Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method].Rev Bras Cir Cardiovasc. 2008; 23: 29-34
- Optimization of Human Peripheral Blood Mononuclear Cells (PBMCs) Cryopreservation.Int J Mol Cell Med. 2012; 1: 88-93
- Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.J Transl Med. 2017; 15: 59
- Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies.J Transl Med. 2014; 12: 241
- Simplified process for the production of anti-CD19-CAR-engineered T cells.Cytotherapy. 2013; 15: 1406-1415
- Cellular Therapy: Principles, Methods, and Regulations.AABB, Bethesda, MD2016
- Controlled incremental filtration: a simplified approach to design and fabrication of high-throughput microfluidic devices for selective enrichment of particles.Lab Chip. 2014; 14: 4496-4505
- A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma.Sci Rep. 2016; 6: 35943
- A portable system for processing donated whole blood into high quality components without centrifugation.PLoS One. 2018; 13e0190827
- Deterministic Lateral Displacement: The Next-Generation CAR T-Cell Processing?.SLAS Technol. 2018; 23: 338-351
- Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation.Sci Rep. 2017; 7: 9915
- Deterministic hydrodynamics: taking blood apart.Proc Natl Acad Sci U S A. 2006; 103: 14779-14784
- Critical particle size for fractionation by deterministic lateral displacement.Lab Chip. 2006; 6: 655-658
- Microfluidic device for label-free measurement of platelet activation.Lab Chip. 2008; 8: 925-931
- Deterministic lateral displacement for particle separation: a review.Lab Chip. 2014; 14: 4139-4158
Article info
Publication history
Published online: January 16, 2019
Accepted:
December 26,
2018
Received:
November 16,
2018
Identification
Copyright
© 2018 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.
