Serum free media SFM chemically defined media A

Serum free media (SFM) – chemically defined media A chemically defined medium is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known. Standard cell culture media are commonly supplemented with animal serum (such as fetal bovine serum, FBS) as a source of nutrients and other ill-defined factors. . . There is a clear distinction between serum-free media and chemically defined media. Serum-free media may contain undefined animal-derived products such as serum albumin (purified from blood), hydrolysates, growth factors, hormones, carrier proteins, and attachment factors. These undefined animal-derived products will contain complex contaminants, such as the lipid content of albumin. In contrast, chemically defined media require that all of the components must be identified and have their exact concentrations known… To achieve this chemically defined media is commonly supplemented with recombinant versions of albumin and growth factors, usually derived from rice or E. coli, or synthetic chemical such as the polymer polyvinyl alcohol which can reproduce some of the functions of BSA/HSA. The constituents of a chemically defined media include: a basal media (such as DMEM, F 12, or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants and energy sources), which is supplemented with recombinant albumin, chemically defined lipids, recombinant insulin and/or zinc, recombinant transferrin or iron, selenium and an antioxidant thiol such as 2 (b)mercaptorthanol or 1 -thioglycerol. • Avoidance of batch to batch variation of bovine serum or albumin, which causes inconsistency in growth-promoting properties. • Elimination of the risk of contaminants—viruses, mycoplasma, prions from animal-derived products which may be transmitted to the end product used by humans, e. g. , bovine spongiform encephalopathy (BSE) or mad cow disease. • Elimination of factors that may interfere with hormones or growth factors when studying their interaction with cells. • Removal of concerns regarding the limited availability of fetal bovine serum, with periods of world shortages. • Reduced cost: fetal calf serum can account for up to 85% of the over-all cost of the medium when calculated for large-scale cultures. • There are increasing concerns about animal suffering inflicted during serum collection that add an ethical…

“Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions” Shuyuan Yao etb al. PNAS 2006 Embryonic stem cells (ESCs), typically derived from the inner cell mass of the blastocyst, can be propagated indefinitely and differentiated into all of the cell types of the embryo properly… Human and mouse ESCs are conventionally maintained in culture with feeder cells and/or mixtures of exogenous factors. However, unknown factors secreted from the feeder cells or contained in the serum may have undesired activities (e. g. , inducing differentiation or cell death). . Such undefined culture conditions is the main source of inconsistency in large scale and long-term expansion of undifferentiated ESCs… Recent studies have shown that feeder-fibroblast conditioned medium (CM), high concentrations of basic FGF (b. FGF) and combinations of b. FGF with. . signaling molecules can support long term culture of h. ESCs grown on an extracellular matrix (ECM)- coated surface (e. g. , Matrigel) in feeder-free conditions. However, these medium conditions typically contain a proprietary serum replacement product, which is a complex mixture of many unknown factors with varying batch-to-batch activities. . The proprietary serum replacement (GIBCO) used widely in h. ESC culture contains strong BMP-like activity that had to be antagonized by the addition of Noggin (500 ng/ml) along with increased concentration of b. FGF (40 ng/ml) for the long term expansion of undifferentiated h. ESCs on Matrigel in the absence of feeder cells or feeder-cell CM… We reasoned that the substitution of the serum replacement with chemically defined components may avoid the use of BMP-antagonizing molecules. To test such hypothesis, we devised a CDM (herein referred as N 2 -CDM) consisting of DMEM/F 12… and 0. 5 mg/ml BSA (faction V). This N 2 -CDM was further supplemented with different concentrations of b. FGF and evaluated for their ability to support self-renewal of undifferentiated h. ESCs grown on Matrigel… 20 ng/ml b. FGF supplemented N 2 -CDM was sufficient for growing . . h. ESCs for 17 passages over 5 months. Under such N 2 -CDM conditions, h. ESCs grow as large compact colonies, similar to undifferentiated h. ESC colonies grown under CM or feeder cell conditions.

Bone morphogenetic proteins (BMPs) are a group of growth factors… Originally discovered by their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the The N 2 supplements (GIBCO) contain 1 m. M human transferrin (Holo), 8. 61 M recombinant human insulin, 1 m. M progesterone, 1 m. M putrescine, and 1 m. M selenite. T Gibco B-27 supplements, first publication (1989) Gregory Brewer et al: “We report that isolated hippocampal neurons from embryonic day 18 rats can be cultured for several weeks at low densities which permits the determination of individual connections. A serum-free medium was modified from the formulation of Romijn to include the biological anti-oxidants vitamin E, glutathione, pyruvate, catalase and superoxide dismutase. Neuronal survival of 80% and neuritogenesis greatly exceeded that seen in serum-based cultures. It appeared that vitamins E, A and linolenic acid promoted neuritogenesis. The beneficial effects of the antioxidants suggested a toxic role of oxygen… The B 27 supplements (GIBCO) contain D-biotin, BSA (fatty acid-free, fraction V), catalase, L-carnitine HCl, corticosterone, ethanolamine HCl, D-galactose (anhydrous), glutathione (reduced), insulin (human, recombinant), linoleic acid, linolenic acid, progesterone, putrescine 2 HCl, sodium selenite, superoxide dismutase, T-3 albumin complex, DL--tocopherol, DL- -tocopherol acetate, and transferrin (human, iron-poor). Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced and marketed by Corning Life Sciences and BD Biosciences… Matrigel resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate (basement membrane matrix) for culturing cells. Teratoma is a tumor with tissue or organ components resembling normal derivatives of more than one germ layer. In this study Three to five million cells were injected under the kidney capsule of nude mice. b. FGF is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by BMP. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems.

Fig. 1. N 2 and N 2/B 27 -CDM plus 20 ng/ml b. FGF support long-term selfrenewal of h. ESCs. (A) Serially passaged H 1 and HSF 6 h. ESCs grown on a Matrigel coated surface in 20 ng/ml b. FGF-supplemented N 2 - CDM (Upper) or N 2/B 27 -CDM (Lower) form large compact colonies. (B) H 1 and HSF 6 h. ESCs cultured in the above conditions show positive staining of Tra-1 -81, Oct 4, Tra-1 -60, and SSEA-4. (C) RT-PCR analysis shows that the serially passaged H 1 h. ESCs cultured in the above CDM conditions express Nanog, Oct 4, and Sox 2 Fig. 2. (A) Karyotype analysis of passage 8 and 22 H 1 -h. ESCs cultured under the feeder-free condition in the N 2/B 27 -CDM plus 20 ng/ml b. FGF using Gbanding. A normal karyotype was observed for all analyzed nuclei (two representative examples are shown). (B) Complex differentiation developed in the h. ESC-derived teratomas 4– 5 weeks after inoculation in nude mice. Typical differentiated tissues are shown. a, Glandular acinusforming epithelium; b, osteoid-forming cells; c, neuroepithelial (ependymal) rosette; d, pigmented retinal epithelium; e, neuroepithelial rosette and glandular-type epithelium; f, columnar epithelium.

Fig. 5. N 2/B 27 -CDM plus activin A and BMP induced marker expression associated with cardiac muscle lineage. (A) Four-day old undifferentiated h. ESC colonies were treated with 50 ng/ml activin A and 50 ngml BMP-4 for 4 days in the N 2/B 27 -CDM and further cultured for an additional 8– 10 days in the basal N 2/B 27 -CDM. (B) Immunostaining shows expression of specific cardiomyocyte markers: MHC, cardiac Troponin I, MEF-2, and GATA-4. (C) RT-PCR shows the expression of MHC, cardiac Troponin I, Nkx-2. 5, and ANF.

“Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design” Min Kyoung Jeon et al, BMC Biotechnology, 2010 …In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs) for adoptive cell therapy, a statistical optimization approach… was adopted. A basal medium was prepared by supplementing RPMI 1640 medium with insulin, albumin (forms the extracellular physical environment, antioxidative properties, carrier function and more), ferric citrate (provide the cells with the vital mineral – iron), ethanolamine (plays a role in cell growth – propagation through cell cycle checkpoints), fatty acids, glutamine (intermediate in many cell synthetic pathways), sodium pyruvate (energy), 2 -mercaptoethanol (antioxidant), 1 -thioglycerol (anti-oxidant), nonessential amino acids, and vitamins (enzyme co-factors & more). In order to further improve the SFM, 4 supplements, phosphatidylcholine (membrane biogenesis), polyamine supplement (Sigma #P 8483 supports protein synthesis), antioxidant supplement (Sigma A 1345), and cholesterol (membrane biogenesis & more), were selected as potential growth enhancers. . transfer of autologous or heterologous therapeutic cells into a patient following an in-vitro manipulation. For example in cancer therapy, t cells are extracted from the patient, genetically modified and cultured in vitro and returned to the same patient…

√ √ √ Figure 1: Growth profiles of cells cultured in 8 different SFM defined by the “fractional factorial design”. Cell cultures were replicated independently with PBMCs prepared from three different donors. This figure shows one representative growth profile among the three cultures. Black circles, SFM #1; white circles, SFM #2; inverted black triangles, SFM #3; white triangles, SFM #4; black squares, SFM #5; white squares, SFM #6; black diamonds, SFM #7; white diamonds, SFM #8. The compositions of the supplements for the eight media are shown in Table 2.

Normal probability plot of the effects obtained from 4 supplements. Black circles, phosphatidylcholine; white circles, polyamine supplement; inverted black triangles, antioxidant supplement; white triangles, cholesterol. The data points were plotted using maximum viable cell concentrations of three independent cultures. Figure 3 A shows various combinations of cholesterol and polyamine supplement with a range of 0 and 4× in these SFM. The results of the analysis of the response surface design using Design. Expert® led to the optimized composition of SFM - 3. 3× of cholesterol (13. 2 mg/l) and 0. 1× of polyamine supplement (Sigma, #P 8483).

(Spot forming cells) Figure 5 Functional assays of CMV-specific CTLs generated from PBMCs cultured in the developed SFM and SCM. A. Cytotoxicity of CMV-specific CTLs generated from PBMCs cultured in the developed SFM (black circles) and SCM (white circles) was tested for their ability to lyse target cells. B. CMV-specific responses obtained by ELISpot using the developed SFM and SCM as culture media. Functional assays were duplicated independently and each experiment was carried out in triplicates. Error bar represents the standard deviation (n = 3). PBMCs cultured in vitro using culture media supplemented with IL-2 … were plated… in a 24 -well culture plate (Nunc) with 2 ml of the corresponding culture media and directly stimulated with peptides at a concentration of 10 μg/ml (day 0) and with peptidepulsed of autologous DCs (at a ratio of 1: 10 DCs to PBMCs… for a 4 - week expansion). Every two days, 1 ml of culture supernatant was gently replaced with 1 ml of the fresh medium supplemented with IL-2 (50 U/ml, Millipore). After 24 days of cultivation, cells were subjected to cytotoxicity and ELISpot assays. ELISpot assay was carried out as described previously. . Polyvinylidene fluoride (PVDF) plates (Millipore) were coated with anti-IFN-g monoclonal antibody… blocking with culture media (SFM or SCM)… peptide-sensitized PBMCs were plated in triplicates with peptide-presenting autologous DCs. . After incubation for 24 h, cells were removed from the plates by 5 washes with PBS… Wells were incubated with 100 μl of biotinylated monoclonal anti-human IFN-g antibody… avidinhorseradish peroxidase conjugate was added. . The visible spots were counted. .