The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens.

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium.

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for (15)N and (13)C,(15)N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.

Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors.

AIMS: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors. METHODS AND RESULTS: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors. CONCLUSIONS: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells.

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l(-1) YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 microg ml(-1), which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q(hTPO) (the specific rate of hTPO production). The supplementation of YH in SFM increased q(hTPO) by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.

An animal origin perspective of common constituents of serum-free medium formulations.

Serum-free formulations may be << re-engineered >> to eliminate traditional protein constituents and replace their biological function with non-protein substitutes. Non-protein additives may also be obtained from animal sources. Nutrient formulations totally free of exogenous protein and containing no materials of animal origin may be designed for high density cell culture and biological production. Cell-culture medium production requires (i) strict vendor qualification and raw material specifications; (ii) scrupulous maintenance of media kitchen facility and equipment; (iii) monitoring of process water; (iv) air-handling systems and technical personnel; (v) clearly-defined manufacturing protocols to ensure correct formulation and dispensing and (vi) validated sanitization processes to guard against cross-contamination within a multi-use facility.

Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line.

The BTI-Tn5B1-4 insect cell line, commercially available as the High Five cell line (Invitrogen), supports higher levels of recombinant protein production compared to existing insect cell lines. Proprietary serum-free media such as ExCell 405 (JRH Biosciences), Express Five (Life Technologies), IS BAC (Irvine Scientific), and CCM3 (HyClone) are available which were developed specifically for a suspension culture of High Five cells. While these media are highly optimized, a lower cost alternative is desirable for large-scale protein production which is also serum-free and supports good cell growth (>5 x 10(6) cells/mL) and recombinant protein production (>50 mg/L of secreted protein). The amino acid and carbohydrate metabolism of the Tn5B1-4 cells was first examined. It was found that asparagine was nearly depleted during batch growth in Ex-Cell 405, without limitations in glutamine, other amino acids, or glucose. Alanine also accumulated to about 35 mM during growth. We then extended the formulation techniques for medium development used for Spodoptera cell lines to the Tn5B1-4 cell line. A medium based on IPL-41 basal medium, Hy-Soy protein hydrolysate (Quest, International), yeastolate ultrafiltrate, a lipid-sterol emulsion, and Pluronic F-68 was developed. Dextran sulfate (100 microg/mL) was used to induce a single cell suspension culture. This medium is denoted as ISYL and performs best when supplemented with a 2.5% lipid-Pluronic F-68 mixture. Supplementation with additional aspargine in a 1.5% lipid-Pluronic F-68 mixture did not improve growth, suggesting that a lipid was growth-limiting and not an amino acid. Ex-Cell 405 and ISYL with 2.5% lipid-Pluronic F-68 supplement supported virtually identical growth rates, extent of growth (ca. 6.0 x 10(6) cells/mL) in an 80% oxygen atmosphere, and supported production of SEAP (secreted human alkaline phosphatase) at a volumetric level of about 65-70 mg/L. Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures.