For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.

Lentiviral vectors have played a critical role in the emergence of gene-modified cell therapies, specifically T cell therapies. Tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta) and most recently brexucabtagene autoleucel (Tecartus) are examples of T cell therapies which are now commercially available for distribution after successfully obtaining EMA and FDA approval for the treatment of blood cancers. All three therapies rely on retroviral vectors to transduce the therapeutic chimeric antigen receptor (CAR) into T lymphocytes. Although these innovations represent promising new therapeutic avenues, major obstacles remain in making them readily available tools for medical care. This article reviews the biological principles as well as the bioprocessing of lentiviral (LV) vectors and adoptive T cell therapy. Clinical and engineering successes, shortcomings and future opportunities are also discussed. The development of Good Manufacturing Practice (GMP)-compliant instruments, technologies and protocols will play an essential role in the development of LV-engineered T cell therapies.

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell ‘detachment’ from the microcarriers’ surface followed by the ‘separation’ of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too

This is an open access article published by Elsevier under the CC BY license\ud (http://creativecommons.org/licenses/by/4.0/)Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells\ud and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the\ud full value of this emerging field can be realized. The characterization of input material for cell-based therapy\ud bioprocesses from multiple donors is necessary to identify and understand the potential implications\ud of input variation on process development. In this work, we have characterized bone marrow derived\ud human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of\ud the measurable input variation on the development of autologous and allogeneic cell-based therapy\ud manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines\ud over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the\ud culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has\ud been demonstrated that this variation results in a range in the process time between these donor hMSC\ud lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing\ud products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT\ud criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the\ud different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of\ud 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent\ud of donor variability throughout the expansion process. Measuring informative product attributes during\ud process development will facilitate progress towards consistent manufacturing processes, a critical step\ud in the translation cell-based therapies

This is an open access article published by Elsevier under the CC BY license\ud (http://creativecommons.org/licenses/by/4.0/).In our recent work in different bioreactors up to 2.5 L in scale, we have successfully cultured hMSCs using\ud the minimum agitator speed required for complete microcarrier suspension, NJS. In addition, we also\ud reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two\ud donors. The essence of the protocol is the use of a short period of intense agitation in the presence of\ud enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than\ud the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective\ud for culture at NJS and detachment in-situ in 15 mL ambrTM bioreactors, 100 mL spinner flasks and 250 mL\ud Dasgip bioreactors. In these experiments, cells from four different donors were used along with two\ud types of microcarrier with and without surface coatings (two types), four different enzymes and three\ud different growth media (with and without serum), a total of 22 different combinations. In all cases after\ud detachment, the cells were shown to retain their desired quality attributes and were able to proliferate.\ud This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range\ud of scales of operation