Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type 1 diabetes, however, it is severely limited by the shortage of organ donors. Ex vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 diabetes. It has recently been shown that, even in the absence of fibrotic over-growth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

A modular approach to study protein adsorption on surface modified hydroxyapatite

Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces.

Proceedings of the Eighteenth Annual Biochemical Engineering Symposium

The eighteenth annual biochemical engineering symposium was held during April 22–23, 1988 at the YMCA of the Rockies conference center in Estes Park, Colorado, under the sponsorship of the University of Colorado. Previous symposia in this series have been hosted by Kansas State University (1st, 3rd, 5th, 9th, 12th, 16th), University of Nebraska-Lincoln (2nd, 4th), Iowa State University (6th, 7th, l0th, 13th, 17th), University of Missouri–Columbia (8th, 14th), and Colorado State University (11th, 15th). Next year's symposium is scheduled to be held at the University of Missouri-Columbia. The symposia are devoted to talks by students about their ongoing research. Because final publication usually takes place elsewhere, the papers included in the proceedings are brief, and often cover work in progress. ContentsApplications of mass spectrometers in biochemical engineeringJohn P. McDonald, Ayush Gupta, and Lourdes Taladriz, Kansas State University Enzymatic hydrolysis of corn gluten proteinsJulie Hardwick; Iowa State University Improved Acetone-Butanol Fermentation AnalysisZ. Buday; Colorado State University On-Line State Identification for Batch FermentationD. A. Gee and W. F. Ramirez; University of Colorado Role of Spargers in Air-Lift ReactorsPeter U. Sohn and Rakesh K. Bajpai; University of Missouri–Columbia The Interaction of Microcarriers and Turbulence within an Airlift FermenterG. Travis Jones; Kansas State University Oxygen Diffusion in the Inter-Fiber Gel/Cell Matrix of NMR-Compatible Hollow Fiber Bio-ReactorsS. L. Hanson, B. E. Dale, and R. J. Gillies; Colorado State University Characterization of Ca-alginate Gel Beads FormationHorngtwu Su, Rakesh K. Bajpai, and George W. Preckshot; University of Missouri–Columbia Metabolic Effects of Chloramphenicol Resistance in the Recombinant Host/Vector System: E. coli RRl [pBR329]William E. Bentley, Dana C. Andersen, Dhinakar S. Kompala, and Robert H. Davis; University of Colorado Genetic Engineering of Beta-Galactosidase to Aid in Fermentation Product Recovery by Polyelectrolyte PrecipitationD. E. Parker, C. E. Glatz, J. Zhao, C. F. Ford, S. M. Gendel, and M. A. Rougvie; Iowa State University Biodegradation of Organic Compounds in SoilLourdes Taladriz, L. E. Erickson, and L. T. Fan; Kansas State University Effect of Dilution, pH and Nutrient Composition on the Biodegradation of Metalworking FluidsAyush Gupta, L. E. Erickson, and L. T. Fan; Kansas State University Dissolved Hydrogen Correlation with Redox Potential in Acetone-Butanol FermentationXiangdong Zhou; Colorado State University Modeling of Ensiling Fermentation of Sweet SorghumA. K. Hilaly; Colorado State University

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

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. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.

Scaffolds for controlled release of cartilage growth factors

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