Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented Bio Med Frontiers with human plasma from group AB donors.
As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture.
Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control).
An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis.
- There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A.
- Compared to controls, Learn more about the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used.
- This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT.
The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.
Screening of Media Supplements for High-Performance Perfusion Cultures by Design of Experiment.
- Perfusion is considered as the preferable unit operation mode for fully integrated continuous bioprocessing. However, the inherent complex process control, long process development times, and lack of suitable scale-down models for high-throughput screening are reasons why perfusion processes are still not routinely applied in cell culture technology.
- Advantages of perfusion are maintenance of a consistent cellular environment, a constant high-quality product flow, enhanced volumetric bioreactor productivity, and small lab footprint.
- Here, we provide guidelines for screening different proprietary but commercially available HyClone™ Cell Boost™ supplements in a Design of Experiment (DoE) approach to spike the HyClone™ CDM4NS0 basal media for enhanced product titers in small-scale TubeSpin models.
- These surrogate semi-perfusion cultures were successfully realized by a daily complete media exchange routine resulting in high viable cell densities for extended time periods at minimal media consumption.
- This technique was leveraged to define the potential of different perfusion media formulations.
Rapid development of clone-specific, high-performing perfusion media from established feed supplements.
- Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry.
- This is due to increased space-time yields (STY) and a short residence time of the recombinant protein in the bioreactor.
- Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone-specific, high-performing perfusion media we present a straightforward and rapid two-step approach combining commercially available basal media and feed supplements using design-of-experiment (DoE).
- First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small-scale semi-continuous perfusion cultures. The final media formulation is supported by statistical response surface modelling of a set of cultivation experiments with blended media formulations.
- Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 106 c/mL within two weeks at minimum cell-specific perfusion rates (CSPR) as low as 10 to 30 pL/c/d. Obtained STYs of 0.4-1.2 g/L/d represent a 10-fold increase compared to batch cultures.
- This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium and established feed solutions. This article is protected by copyright. All rights reserved.
Establishment of an in vitro model of cultured viable human, porcine and canine skin and comparison of different media supplements.
Transdermal drug delivery provides several advantages over conventional drug administration, such as the avoidance of first-pass metabolism and better patient compliance.
In vitro research can abbreviate and facilitate the pharmaceutical development considerably compared to in vivo research as drug screening and clinical studies can be reduced.
These advantages led to the development of corresponding skin models. Viable skin models are more useful than non-viable ones, due to the influence of skin metabolism on the results. While most in vitro studies concentrate on evaluating human-based models, the current study is designed for the investigation of both human and animal diseases.
So far, there is little information available in the literature about viable animal skin cultures which are in fact intended for application in the veterinary and not the human field. Hence, the current study aims to fill the gap. For the in vitro viable skin model, specimens of human, porcine and canine skin were cultured over two weeks under serum-free conditions.
To evaluate the influence of medium supplementation on skin viability, two different supplement mixtures were compared with basic medium.
The skin specimens were maintained at a viability-level >50% until the end of the study.
From the tested supplements, the addition of bovine pituitary extract and epidermal growth factor increased skin viability whereas hydrocortisone and insulin induced a decrease.
This in vitro viable skin model may be a useful tool for the investigation of skin diseases, especially for the veterinary field.
Human Platelet Lysate Media Supplement Supports Lentiviral Transduction and Expansion of Human T Lymphocytes While Maintaining Memory Phenotype.
Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies.
The production of CAR T cells usually involves viral transduction and ex vivo culture of T cells.
The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production.
For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters.
Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days.
T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7.
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The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance in vivo performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.