Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient non-enzymatic cell harvesting during 3D culturing

Enzymatic detachment of cells might damage important features of cells and could affect subsequent function of cells in various applications. Therefore, non-enzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, we synthesized thermosensitive PNIPAm-co-AAc-b-PS and PNIPAm-co-AAm-b-PS copolymers and LCST was tuned near to body temperature. Then, polymer solutions (5% w/v, 10% w/v, and 20% w/v) were spin coated to prepare films for cell adhesion and thermal-induced cell detachment. The apha-step analysis and SEM image of the films suggested that the thickness of the films depends on the molecular weight and concentration which ranged from 206 nm to 1330 nm for PNIPAm-co-AAc-b-PS and 97.5 nm to 497 nm for PNIPAm-co-AAm-b-PS. The contact angles of the films verified that the polymer surface was moderately hydrophilic at 37°C. From cell attachment and detachment studies, RAW264.7 cells, were convincingly proliferated on the films to a confluent of >80 % within 48 days. However, relatively more cells were grown on PNIPAm-co-AAm-b-PS (5%w/v) films and thermal-induced cell detachment was more abundant in this formulation. As a result, commercial cytodex 3 microcarrier was coated with PNIPAm-co-AAm-b-PS (5%w/v) and interestingly enhanced cell detachment with preserved potential of recovery was observed at low temperature during 3D culturing. Thus, surface modification of microcarriers with PNIPAm-co-AAm-b-PS could be vital strategy for non-enzymatic cell dissociation and able to achieve adequate number of cells with maximum cell viability, and functionality for various cell-based applications. Keywords: surface coated microcarriers; thermosensitive polymer; non-enzymatic cell detachment

Production of Adult Human Synovial Fluid-Derived Mesenchymal Stem Cells in Stirred-Suspension Culture

The chondrogenic potential of synovial fluid-derived mesenchymal stem cells (SF-MSCs) supports their use in cartilage regeneration strategies. However, their paucity in synovial fluid necessitates their proliferation in culture to generate clinically relevant quantities. Here it was determined that 125 mL stirred suspension bioreactors utilizing Cytodex-3 microcarrier beads represent a viable platform for the proliferation of these cells. During the inoculation phase, a bead loading of 2 g/L, an inoculation ratio of 4.5 cells/bead, and continuous agitation at 40 rpm in a medium with 5% serum resulted in high cell attachment efficiencies and a subsequent overall cell fold expansion of 5.7 over 8 days. During the subsequent growth phase, periodic addition of new microcarriers and fresh medium increased culture longevity, resulting in a 21.3 cell fold increase over 18 days in the same vessel without compromising the defining characteristics of the cells. Compared to static tissue culture flasks, a bioreactor-based bioprocess requires fewer handling steps, is more readily scalable, and for the same cell production level, has a lower operating cost as it uses approximately half the medium. Therefore, stirred suspension bioreactors incorporating microcarrier technology represent a viable and more efficient platform than tissue culture flasks for the generation of SF-MSCs in culture.

Expansion and Harvesting of hMSC-TERT

The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d-1. The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes.

Techniques for Measurement of Oxygen Consumption Rates of Hepatocytes during Attachment and Post-Attachment

Three techniques for measuring oxygen consumption rate (OCR) of cultured cells relevant to the development of bioartificial liver devices are reported. In an oxystat apparatus, HepG2 cells immobilised on Cytodex 3 microcarriers at a concentration of 106 cells ml-1 had a mean OCR of 0.7 nmol s-1/106 cells. The OCR decreased with increasing cell density, a characteristic previously reported for other cell lines. Rat hepatocytes immobilised on single collagen layers in a flow cell and challenged with ammonia had a mean OCR of 0.59 nmol s-1/106 cells. A novel two-compartment oxystat system was used to determine the OCR of rat hepatocytes during the attachment phase. OCR declined from 1.0 nmol s-1/106 immediately after seeding to 0.7 nmol s-1/106 cells at nine hours. The low OCR for HepG2 reflects loss of certain oxygen dependent metabolic pathways. The OCR measured for rat hepatocytes during and post-attachment are significantly higher than those reported elsewhere and have major implications for the development of bioartificial liver devices.