Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors – III

[Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors - I & II]

2.4. Harvest

Once the bioreactor control system and optimized medium feeding strategy have proven to effectively expand hMSCs, the next step is to determine the optimal time to harvest these cells as the final product. Compared with planar culture, microcarrier-based culture faces challenges in hMSC harvesting. At the time of harvest, hMSCs typically occupy most of the microcarriers, and the degree of microcarrier aggregation is often very high, potentially further increasing the difficulty of cell harvesting. Procedures based on planar culture can be used as a guide.The entire harvest process of microcarrier culture begins with the separation of cells from the microcarriers. This includes steps such as draining the medium, adding PBS and washing with PBS, draining the PBS, adding dissociation buffer for stirring and incubation, and quenching with medium.Then, the following steps are included to effectively remove microcarriers and concentrate large quantities of cell suspension for subsequent downstream bioprocessing. Each step of removing liquid from the culture system inevitably leads to some cell loss. For instance, draining the medium or wash buffer may also remove some microcarriers with adherent hMSCs, and both the microcarrier removal and cell suspension/concentration steps involve some cell loss. Separation efficiency may be another factor reducing the total cell yield. Therefore, many methods have been developed to improve hMSC harvesting efficiency.

The most critical part of harvest efficiency is the cell detachment process. Nienow et al. reported a combination of 21 in-situ detachments using dissociation reagents Trypsin-EDTA, TrypLE Express, and Accutase in 15 mL ambr and 100 mL DASGIP bioreactors as well as 100 mL roller bottles to optimize the detachment efficiency in hMSC microcarrier cultures. Essentially, their protocol combines chemical reactions with physical forces required for maximum detachment efficiency through short periods of intense agitation with dissociation reagents. At any time point, it can be checked under a microscope whether microcarriers are still attached to cells by visual inspection of the sample. Moreover, once cells are detached into single cells or small clumps smaller than the Kolmogorov turbulence scale, they will no longer be affected by fluid shear stress. After cell detachment, microcarriers need to be removed to obtain a pure cell suspension. Sterile sieves can be used to filter out microcarriers. For accommodating large-scale preparations, disposable filtration bags are commercially available. For example, Thermo Fisher Scientific’s Harvestainer™ bioprocess container is designed to separate microcarrier beads from harvested culture broth in closed systems of various sizes, including 3 L, 12 L, 25 L, and 50 L, which should cover most scales formulated to date. This involves simply assembling a micro-screen bag inside a sterile containment bag to allow microcarriers to be captured in the screen bag while detached cells pass through. EMD Millipore’s OptiCap® XL 1 capsule filter also provides effective filtration to separate cells from microcarriers. The mesh size must be between the diameter of the microcarrier and the cell, ranging from 60 to 100 µm.

Regardless of the method used, the harvesting process involves several complex steps that must be carried out promptly.To reduce complexity, innovative types of microcarriers have been developed to simplify procedures and enhance production speed and efficiency. Soluble microcarriers represent an innovative approach that allows for the easy recovery of all cells without the need for further filtration and have been used in scaled-up bioreactor cultures.Thermo-responsive microcarriers also underwent thermal lift harvesting tests. Despite these advances, there is still room for further improvement to accelerate the harvesting process.

After removing microcarriers, the clarified hMSC suspension typically has a larger volume (>20 L) compared to comparable planar cultures. Laboratory-scale centrifuges lack the capacity to handle such large volumes. State-of-the-art continuous flow technologies have been developed for efficient cell concentration. Tangential flow filtration, also known as crossflow filtration, is a design that enhances permeate flow rates by setting the flow direction perpendicular to the filtration direction, with many commercial products available. Another design is countercurrent continuous centrifugation. Due to the balance of fluid flow opposing the centrifugal force, these systems can retain cells in a fluidized bed. These methods accelerate the processing time for large-scale cell concentration and reduce the exposure time of cells to centrifugal forces but still require further optimization to achieve maximum hMSC yield.

3. Sampling and Cell Counting

The efficacy of these cells as therapeutic agents depends on the ability to obtain a large quantity of healthy and viable hMSCs. Therefore, an accurate and consistent method for counting suspended cells is crucial. Additionally, cell counting in microcarrier-based bioreactor cultures becomes more challenging due to interference from microcarriers and their inherent aggregation in the culture. Compared to the consumption of nutrients and accumulation of waste, determining cell numbers is considered the most direct evidence supporting and confirming successful cell expansion, thus also guiding decisions and directions for future process development. Essentially, cell counting serves as a key reference throughout the entire biological process. Before counting the cells, it is essential to collect a representative sample. Factors such as the position of the sampling port, stirring speed during sampling, and the process of handling the sample can all potentially alter the counting results. Optimization of the sampling method is the first step, especially in the later stages of cell culture when hMSCs grow in multi-microcarrier aggregates, which may present challenges for sampling optimization.

Cell counting is a relatively simple process, and many methods have been established to obtain homogeneous cell suspensions from monolayer cultures. This method involves counting the number of cells within a fixed volume, then converting the count to sample size, and further extrapolating it to the entire culture system.

Generally speaking,Cell Counting Methods in Microcarrier Culture: (1) Cell counting after cells detach from microcarriers; (2) Cell counting after microcarrier dissolution; (3) Nuclei counting after cell lysis on microcarriers; (4) DNA quantification; (5) Metabolic assays.A counting method with simple steps and high throughput would be an ideal choice and more advantageous than assay-based methods. Moreover, since dissolvable microcarriers are not always feasible, detaching and lysing cells from microcarriers is often a more suitable option.

In addition to manual cell counting using a hemocytometer, automated cell counters have been widely adopted in hMSC production for the rapid measurement of cell numbers in replicate samples. Although the principle simply involves counting all cells and determining viability based on membrane integrity, different automated cell counters may yield varying results. Trypan blue enhances cell counting through viability information, while fluorescent dyes such as acridine orange, calcein, DAPI, propidium iodide, etc., can provide higher color contrast for counting live and dead cells via automated cell and nucleus counters, with or without lysis buffer. These automated counters primarily analyze images of stained cells/nuclei and calculate particle counts. Importantly, gates should be set according to the size of cells/nuclei to exclude irrelevant cell debris or fragmented nuclei that may be present in the sample. The typical validated range for automated counters is 10^4 to 10^7, as this provides sufficient particles for counting without overcrowding, which might prevent the device from identifying individual particles. Notably, when counting cells cultured on microcarriers, automated cell counters suitable for planar cultures may be less efficient due to the risk of microcarriers clogging the loading tool.

4. Conclusion

Our previous research reviewed and summarized the changes in hMSC therapeutic efficacy caused by microenvironmental changes when switching from planar culture to microcarriers, finding a significant increase in the differentiation potential of osteogenic and chondrogenic lineages, while a notable decrease in the differentiation potential of adipogenic lineage. Improvements in migration ability, anti-inflammatory, and immunomodulatory capacities were also observed.We also acknowledge the drawbacks of microcarrier cultures, primarily the heterogeneity caused by discontinuous growth surfaces and uneven shear stress distribution within bioreactors. Nevertheless, despite these limitations, microcarrier-based bioreactor systems remain one of the most promising and profitable methods for the production of hMSC-based products for commercialization.This review focuses on the development of microcarrier-based bioreactors, which have great potential in the scale-up process. We also discuss the required changes in bioprocessing, including seed culture, inoculation, amplification, harvesting, and cell counting necessary for microcarrier cultures. Additionally, bioreactor parameters such as temperature, pH, dissolved oxygen, mixing, and feeding are summarized. Finally, we detail various cell counting methods applied to hMSC microcarrier cultures.

A.Tsai, C.A.Pacak, Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors. Bioengineering, 2021.