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Background: In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined., Methods: We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay., Results: We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 10 3 cells cm -2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields., Conclusions: Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The discovery of the genome-editing tool CRISPR-Cas9 is revolutionizing the world of gene therapy and will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is an efficient and effective transfer of the nucleic acids carrying the relevant information to their target destination, the nucleus of the cells. Baculoviruses have shown to be very suitable transport vehicles for this task due to, inter alia, their ability to transduce mammalian/human cells without being pathogenic. This property allows the usage of baculovirus-transduced cells as cell therapy products, thus, combining the advantages of gene and cell therapy. To make such pharmaceuticals available for patients, a successful production and purification is necessary. In this chapter, we describe the generation of a pseudotyped baculovirus vector, followed by downstream processing using depth and tangential-flow filtration. This vector is used subsequently to transduce human mesenchymal stem cells. The production of the cells and the subsequent transduction process are illustrated.

Human mesenchymal stem cells (hMSCs) are the current workhorses of regenerative medicine and gene therapy. The corresponding vectors are usually based on lentiviruses, adenoviruses, retroviruses or adeno-associated viruses, but recently they have been joined by baculoviruses, which are more widely known for their role in the development of pesticides and vaccines. Here we show that gene transfer to an immortalized human mesenchymal stroma cell line can be achieved by baculovirus transduction. We also compared the performance of five different constitutive promoters controlling GFP expression. The human elongation factor 1α promoter (EF1α) achieved the strongest GFP expression, whereas the mouse phosphoglycerate kinase 1 promoter (PGK) was the weakest. The human EF1α promoter is therefore recommended for the regulation of genes introduced into hMSC-TERTs by baculovirus vectors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

For cell therapy, a high biomass of human mesenchymal stem cells (hMSCs) is required for clinical applications, such as in the form of encapsulated implants. An easy and reproducible microcarrier-based stirred tank reactor cultivation process for hMSCs in 1.68 L scale is described. To avoid medium changes, studies comparing high-glucose DMEM (DMEM-HG) with low-glucose EMEM were performed showing that high-glucose medium has positive effects on cell proliferation and that cell differentiability remains. Studies on the inoculation strategy and cell density, carrier concentration, volume, and stirrer speed were performed and resulted in a set of optimized parameters, inoculation strategy was found to be 45 minutes of static state and 2 minutes of stirring repeated in 4 cycles. The inoculation density was chosen to be 7×10³ cells/cm2, and the carrier concentration of glass surface carrier was 25 g/L. For the described reactor system, a stirrer speed of 120 rpm for the inoculation process and a daily increase of 10 rpm up to 160 rpm were found to be suitable. Process reproducibility was shown by 3 repeated cultivations at the determined set of parameters allowing high biomass values of up to 7×10⁸ cells per batch. With DMEM-HG, no limitation of glucose was found, and lactate and ammonia remained lower than critical inhibitory concentrations. Comparison of the static (T-flask) and dynamic cultures in the stirred tank reactor showed for both cases, that cells were of high vitality and both maintained differentiability. In both cases, encapsulation of the cells resulted in high bead vitality, a basic requirement for cell therapy application.

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy. [ABSTRACT FROM AUTHOR]

The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types. [ABSTRACT FROM AUTHOR]

The great properties of human mesenchymal stromal cells (hMSCs) make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved. [ABSTRACT FROM AUTHOR]

Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02–2.6 W m −3 ) on the mixing time (t m ). Whereas t m was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30–70 rpm. A high microcarrier concentration (30 g L −1 ) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in t m and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on t m became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m −3 was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date. [ABSTRACT FROM AUTHOR]

The production of stem cells for clinical applications requires a suitable mass expansion and harvest process, which is implemented in a microcarrier-based bioreactor. Important parameters to consider include the choice of microcarriers for cell expansion, the growth medium and the scale-up strategy. We investigated six different microcarriers to determine whether they can support the growth and harvest of primary human mesenchymal stromal/stem cells (hMSCs) derived from bone marrow, and the immortalized cell line hMSC-TERT. A serum-containing medium (SCM) and a new chemically defined medium (CDM) were compared under dynamic culture conditions. We also investigated bead-to-bead transfer from spinner flasks to a stirred tank reactor as a scale-up strategy. We observed cell type-dependent differences in growth rate and attachment behavior on each microcarrier. Missing serum components in the CDM led to slower cell attachment and growth, whereas microcarriers suitable for the SCM were also suitable for CDM. Glass-coated microcarriers supported hMSC-TERT growth and bead-to-bead transfer in SCM, whereas plasma-treated plastic surfaces promoted cell growth in CDM. We demonstrated that stem cell cultures can be scaled up by bead-to-bead transfer, avoiding the need for pre-cultures in tissue flasks. The replacement of pre-cultures with a monitored bioprocess could therefore facilitate the development of conditions suitable for large-scale stem cell production. [ABSTRACT FROM AUTHOR]

Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02–2.6 W m −3 ) on the mixing time (t m ). Whereas t m was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30–70 rpm. A high microcarrier concentration (30 g L −1 ) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in t m and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on t m became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m −3 was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date. [ABSTRACT FROM AUTHOR]