A consensus introduction to serum replacements and serum-free media for cellular therapies.

The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.

Serum starvation: caveat emptor.

Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.

Standardized, defined serum-free culture of a human skin equivalent on fibroblast-populated collagen scaffold.

The defined, serum-free media used in the cultivation of skin equivalents are liable to inter-laboratory variations, require the preparation of multiple additives, and are potentially difficult to replicate. In this study, we assessed the use of standardized, serum-free and bovine pituitary extract-free keratinocyte culture media in the development of a human skin equivalent. After culture at the air-liquid interface for 3 weeks on a fibroblast-populated collagen lattice, an orthokeratinized, pluristratified epithelium was produced which expressed cytokeratins, cornified cell envelope precursors (involucrin, transglutaminase 1, filaggrin) and desmosomal components (desmoglein and desmocollin 1 and 3, plakophilin 1) in a differentiation-specific manner. There was also evidence of basement membrane reconstitution with collagen IV/VII, laminin 5, α6 and ß4 integrin subunit expression at the epithelial-matrix junction. Overall, our findings demonstrate that readily available, defined organotypic culture media can be used to generate a reproducible skin equivalent with hallmarks of epidermal differentiation.

A survey of scientists' awareness of and attitudes to the use of human blood products and alternatives in human assisted reproductive technology.

Scientists working in assisted reproduction [members of Scientists in Reproductive Technology (SIRT) Australia, and subscribers of the online forums EmbryoMail and Quartec] were invited to complete an online questionnaire on the use of human blood products in assisted reproductive technologies (ART). A total of 260 started the questionnaire, with 208 (80%) completing it. A total of 62% of respondents had worked in human ART ≥8 years and 68% had post-graduate qualifications. The majority (82%) reported using products of animal or human origin, with 75% knowing why protein was added to culture media and 41% not worried by this. Almost half (49%) of respondents were unaware of regulations surrounding the use of human blood products in health care and 70% were unaware of adverse events involving human blood products in human ART. Most respondents (70%) indicated that they were not concerned about infections such as hepatitis, but agents such as prions were a cause for concern (57%). A total of 57% of respondents were unaware of alternatives, but 77% would use a suitable alternative. Using blood products in human ART is surrounded by a lack of awareness, often independent of respondents' qualifications or experience. A better understanding of these products and possible alternatives is required if informed decisions about their suitability are to be made.

Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Standardization and comparison of endogenous erythroid colony assays performed with bone marrow or blood progenitors for the diagnosis of polycythemia vera.

The reliability of the assay of endogenous erythroid colony (EEC) formation in serum-free, cytokine-free collagen-based media was investigated in a multicentric study including 140 patients with polyglobuly (80 polycythemia vera (PV), 54 secondary erythrocytosis (SE), six idiopathic erythrocytosis (IE)) and 10 healthy donors. In each center, EEC assays were performed in parallel with progenitor cells from bone marrow (BM) and peripheral blood (PB); two commercialized media and 'low' and 'high' cell plating densities were tested. Negativity of EEC assays was considered certain only when sufficient BFU-E growth was obtained in control cultures with cytokines. In the two media, EEC formation was specific - never observed in cultures of healthy donors or SE patients - and comparable. BM EEC assays were positive (presence of eythroid colonies) for 75% ('low' plating) to 100% ('high' plating) of PV patients; PB EEC assays were positive for 83.3% ('low' plating) to 93.7% ('high' plating) of PV patients (differences not significant). Depending on the medium, 86.2-93.7% of patients with a positive BM EEC assay had a positive PB EEC assay. Hence, a standardized collagen-based EEC assay can be performed with either BM or PB progenitors; the EEC assay described here is positive for at least 75% of PV patients when a single EEC assay is performed, and for at least 94% of PV patients when both BM and PB EEC assays are performed.

The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.

Fetal bovine serum (FBS) is a common supplement to in vitro culture media. A workshop was organized to discuss whether or not fetuses might suffer when blood is withdrawn, and to discuss serum replacement methods. When bovine fetuses are exposed after slaughter of the dam, they can suffer only if they inflate their lungs with air and increase their blood oxygen to levels compatible with awareness. Preventing fetuses from breathing air or killing them by an efficient method, according to clearly defined safeguards, ensures that fetal blood collection is humane. Since serum is a supplement of unknown composition, which could be contaminated with unwanted factors, there are scientific and safety reasons for omitting FBS from culture media. Several media have been developed in which minimal or no animal derived components are present. Also, different cell types have been adapted to serum-free media. As yet, no standard serum free media are present, and each cell type requires its own medium composition. Among other recommendations, the establishment of a public database with information on cell types and their serum-free medium composition is proposed.

Development of cultivation technique for pure Plasmodium falciparum gametocytes.

Pure gametocyte culture of Plasmodium falciparum, isolate KT3, from Kanchanaburi Province, Thailand, was successfully established by 5% sorbitol treatment on days 9, 10 and 11 following initiation of culture. Using medium supplemented with 15% human plasma and activated erythrocytes, daily medium change was not required during the cultivation. There was 99% reduction in the numbers of asexual parasites in the culture but the numbers of gametocytes were not affected. Furthermore, the gametocytes could undergo their usual morphological development with retention of function as demonstrated by the appearance of exflagellating microgametocytes, macrogametocytes and of oocyst formation in midgut of infected mosquito.

Anti-dengue IgG detection by an indirect ELISA.

Protein-free culture media were originally developed for hybridomas to simplify downstream processing and purification. For the same reasons, we have used these protein-free media for passaging dengue 2 virus in C6/36 cells. This provided us with an infected supernatant (DenPF) which could then be used as coating antigens for an indirect enzyme-linked immunosorbent assay (ELISA) to determine dengue IgG levels. Using this preparation, the main immunogenic band as seen by immunoblot appeared to be viral envelope protein (E). Without the high concentrations of "competing" proteins from fetal calf serum (FCS), the Den2PF could be directly coated onto 96-well ELISA plates. The amount of viral proteins in Den2PF appeared to be sufficient so that there was no need for further purification steps, eg polyethylene glycol (PEG) precipitation, which made this preparation cost effective. It compared favorably with the dengue dot enzyme immunoassay (DEIA; sensitivity of 95.7% and specificity of 95.2%).

Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media.

The aim of this study is to improve the cultivation of Leishmania promastigotes without the use of common, semisolid culture media such as Evans' modified Tobie's medium (EMTM), liquid RPMI 1640, and Peptone-yeast extract medium (P-Y). Although EMTM medium permits the growth of a high number of parasites, it is technically difficult to prepare as it requires the use of fresh rabbit blood from animals bred on farms, while RPMI 1640 and P-Y show lower growth rates than the EMTM. There is, therefore, a need to develop new blood-free and time-saving culture systems. The aim of this paper is to propose new modified microbiological media, named RPMI-PY and Tobie-PY, to isolate Leishmania and cultivate parasites for research and diagnostic purposes. This study compares classic culture media to the new media, RPMI-PY and Tobie-PY, and demonstrates that the new media have superior performance in terms of time and parasitic load. The growth rate of the parasite was significantly higher at 24, 48, and 72 hr cultivation, based on counts using Bürker's chambers, when compared to classic media. This study was carried out at the National References Centre for Leishmaniasis (C.Re.Na.L.) where the isolation procedures are conducted daily from a number of different biological matrices.

Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells.

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Development of standardized cell culture conditions for tumor cells with potential clinical application.

BACKGROUND: Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. METHODS: To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. RESULTS: The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. DISCUSSION: We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.

The development, benefits and disadvantages of serum-free media.

Serum-free culture is used routinely for many cell types, especially if transformed, e.g. CHO, hybridoma and recombinant myeloma cell lines. Serum-free medium reduces operating costs and process variability, and removes a potential source of infectious agents. The removal of the components of serum and the elimination of other animal-derived raw materials are proving more challenging. Nevertheless, careful risk/benefit analyses to target R&D toward critical materials is presenting several solutions in this area. A risk/benefit analysis is required for each component replacing serum or serum derivatives. Specific serum proteins used in serum-free media can be eliminated. At present large scale protein-free processes are not routine.

Safety issues of animal products used in serum-free media.

The development of media free of serum and animal or human protein is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. The main drawback associated with the use of serum or animal-derived substances for animal cell technology is the potential introduction of contaminants (adventitious agents) into the process and thus potentially into the final product. This fact led to an increased effort to replace serum-containing with serum-free media. In most cases, these media are supplemented with purified proteins, peptones, or hydrolysates, mainly of animal or human origin. Although such serum-free media are more defined than serum-containing media, the risk of the introduction of viruses by using animal-derived substances is still present, signifying that only a complete replacement of animal-derived substances by non-animal-derived products leads to a relatively safe serum-free medium. The potential replacement of these animal/human-derived substances by those of non-animal origin (e.g. plant origin) will be discussed. In several examples, the potential of serum-free media free of any animal-derived component in supporting cell growth and production of biologicals will be presented. In this context, the risk of using non-animal-derived substances in serum-free media for animal cell technology will be discussed with respect to classically used cell culture media.

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.

[Effect of the supernatant from mice liver cell primary culture on the in vitro growth of Plasmodium falciparum wild isolates]. / Effet du surnageant de culture primaire d'hépatocytes de souris sur la prolifération in vitro des isolats sauvages de Plasmodium falciparum.

Cultivation of Plasmodium falciparum has been a major research success, leading to a greater understanding of the parasite. Despite the fact that several P. falciparum clones have been maintained in continuous culture in different laboratories, research in genomics and proteomics would require parasitic material produced from fresh wild isolates. We have tested the effect of the supernatant from primary culture of mice hepatocytes on in vitro growth of P. falciparum isolates. Parasitized blood samples were collected from Madagascan malarious patients naturally infected. Isolates proliferation was assessed by use of isotopic method. The asexual erythrocytic stages of P. falciparum were grown for 42 hours in RPMI 1640-based medium plus L15 medium-based supernatant from mice liver cells culture, and in standard RPMI 1640-based medium alone. The mean of parasite growth was 1.5 times greater when the standard medium was enriched with the liver cells layer supernatant at a proportion of 10% and 15% (v/v). The usefulness of P. falciparum ex-vivo culture and of the hepatocytes in vitro primary culture is discussed.

Serum-free culture of human megakaryocyte progenitors.

A serum-free method for the culture of megakaryocyte progenitor cells (burst-forming units megakaryocyte, BFU-Mk) from human bone marrow is described, using recombinant cytokines (interleukin[IL]-3, IL-6, IL-11 and stem cell factor [SCF]) to provide megakaryocyte colony-stimulating activity. IL-3 alone, but not IL-6, IL-11 or SCF alone, promoted BFU-Mk formation. The combination of IL-3 with IL-11 or with IL-6+SCF was necessary for optimal maturation of the megakaryocytes. No significant difference in BFU-Mk numbers was found between the combinations of IL-3+IL-11 and IL-3+IL-6+SCF. The development of BFU-Mk was found to be maximal at 12-14 d. Using these conditions, normal ranges for human bone marrow BFU-Mk were derived which may provide the basis for studying megakaryocyte colony growth in pathological conditions.

Insulin enhances epidermal growth factor- and transforming growth factor-alpha-stimulated growth in primary cultures of guinea pig gastric mucous epithelial cells.

The effect of insulin on epidermal growth factor (EGF)- and transforming growth factor-alpha (TGF alpha)-induced cell proliferation was examined in primary cultures of gastric surface mucous epithelial cells by using changes in cell counts and DNA as indices of cell growth. With 1.0% fetal bovine serum and Dulbecco's modified Eagle medium (low-serum DMEM) there was no effect of bovine insulin (0.1-10.0 micrograms/ml) on cell growth. Higher doses of insulin (20.0 micrograms/ml) in low-serum DMEM were able to stimulate growth of these cells compared with controls. In low-serum DMEM with no insulin the lowest effective dose of EGF or TGF alpha for stimulating cell growth was 10 nM and 5 nM, respectively. In low-serum DMEM containing 2.5 micrograms/ml insulin there was a dose-dependent increase in cell growth with EGF and TFG alpha at concentrations of 0.5-10.0 nM. The addition of insulin alone at 2.5 micrograms/ml, EGF alone at 2 nM, or TGF alpha alone at 2 nM in serum-free DMEM media had no effect on gastric surface mucous cell DNA. In serum-free DMEM media with 2.5 micrograms/ml insulin, the addition of 2 nM EGF or 2 nM TGF alpha caused an increase in both cell counts and DNA of 1.80- and 3.50-fold, respectively. The present study demonstrates that insulin, at concentrations that are not mitogenic, will enhance the proliferative effects of EGF and TGF alpha on the growth of primary cultures of guinea pig gastric surface mucous epithelial cells.

Establishment of serum-free pre-colony forming unit assays for differentiation of primitive hematopoietic progenitors: serum induces early macrophage differentiation and inhibits early erythroid differentiation of CD34++CD38- cells.

In this report we show that serum has differentiation-inducing effects on primitive hematopoietic progenitor cells with the CD34++CD38- immunophenotype. Using the pre-colony forming unit (pre-CFU) assay as a model for early myelopoiesis, we compared the effects of serum-containing and serum-free media and evaluated different cytokine cocktails [interleukin (IL)-1, IL-3, IL-6, kit ligand with and without the Flt3/Flk2 ligand (FL)]. In this assay, pre-CFUs are defined as cells unable to form colonies when plated directly in semi-solid assays, but which can differentiate into CFUs when cultured in liquid medium containing early-acting cytokines. In one of the investigated serum-free media, the average myeloid expansion in liquid medium reached up to more than 50% of that obtained in serum-containing medium. In addition, our experiments revealed differences in the clonogenic output between cells cultured in serum-free medium and those cultured in serum-containing medium, demonstrating that serum has a monocyte differentiation-inducing effect on primitive hematopoietic progenitors. Also in serum-free medium, higher proportions of erythroid progenitors were generated. These differentiation-inducing effects of serum further emphasize the need for serum-free culture protocols for hematopoietic graft engineering. Addition of FL to the culture media ameliorated cellular expansion and resulted in a decrease in the proportion of erythroid and granulocyte progenitors and an increase in the proportion of monocyte progenitors. In conclusion, this study shows that good serum-free conditions are available for differentiation assays with primitive hematopoietic progenitors and demonstrates that serum and FL have biasing effects on the initial phase of hematopoietic differentiation, favoring the monocyte lineage.