Animal-free Medium Research Articles

Empirical economic analysis shows cost-effective continuous manufacturing of cultivated chicken using animal-free medium.

Cellular agriculture aims to meet the growing demand for animal products. However, current production technologies result in low yields, leading to economic projections that prohibit cultivated meat scalability. Here we use tangential flow filtration for continuous manufacturing of cultivated meat to produce biomass of up to 130 × 106 cells per ml, corresponding to yields of 43% w/v and multiple harvests for over 20 days. Continuous manufacturing was carried out in an animal-component-free culture medium for US$0.63 l-1 that supports the long-term, high density culture of chicken cells. Using this empirical data, we conducted a techno-economic analysis for a theoretical production facility of 50,000 l, showing that the cost of cultivated chicken can drop to within the range of organic chicken at US$6.2 lb-1 by using perfusion technology. Whereas other variables would also affect actual market prices, continuous manufacturing can offer cost reductions for scaling up cultivated meat production.

Recombinant T7 RNA polymerase production using ClearColi BL21(DE3) and animal-free media for in vitro transcription.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is ~50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics. KEY POINTS: • Optimized IVT system maximizes mRNA yields, enabling the synthesis of long RNAs. • Novel production method yields endotoxin-free and animal-free T7 RNAP. • The T7 RNAP has equivalent specifications and function to conventional T7 RNAP.

Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells.

The use of fetal bovine serum (FBS) in animal cell culture media is widely spread since it provides a broad spectrum of molecules that are known to support cell attachment and growth. However, the harvest and collection procedures of FBS raise ethical concerns and serum is an ill-defined and expensive component. This is especially problematic when it comes to regulatory approval for food applications like cultured meat. The aim of this study is to develop a chemically defined, cost efficient serum-free and animal-free medium that supports the attachment and expansion of bovine myoblasts while maintaining their differentiation capacity. Bovine satellite cells were harvested and isolated from a fresh sample of skeletal muscle tissue and cultured in planar systems. The efficacy of the tested formulations was assessed with metabolic assays and cell counting techniques. Optical microscopy was used to observe cellular morphology and statistical analysis was applied. Based on a comprehensive literature analysis, a defined serum-free medium (SFM) composition was developed consisting of DMEM/F12 as basal medium, supplemented with L-ascorbic acid 2-phosphate, fibronectin, hydrocortisone, GlutaMAX™, albumin, ITS-X, hIL-6, α-linolenic acid, and growth factors such as FGF-2, VEGF, IGF-1, HGF, and PDGF-BB. To our knowledge, this is the first defined serum-free and animal free medium formulation specific for bovine myoblasts to date. We conclude that the SFM formulation supported exponential cell growth up to 97% of the serum—containing golden standard growth medium. All reagents used in this study are chemically defined.

Optimizing large‐scale autologous human keratinocyte sheets for major burns—Toward an animal‐free production and a more accessible clinical application

Background and AimsAutologous keratinocyte sheets constitute an important component of the burn wound treatment toolbox available to a surgeon and can be considered a life‐saving procedure for patients with severe burns over 50% of their total body surface area. Large‐scale keratinocyte sheet cultivation still fundamentally relies on the use of animal components such as inactivated murine 3T3 fibroblasts as feeders, animal‐derived enzymes such as trypsin, as well as media components such as fetal bovine serum (FBS). This study was therefore aimed to optimize autologous keratinocyte sheets by comparing various alternatives to critical components in their production.MethodsHuman skin samples were retrieved from remnant operative tissues. Cell isolation efficiency and viability were investigated by comparing the efficacy of porcine‐derived trypsin and animal‐free enzymes (Accutase and TrypLESelect). The subsequent expansion of the cells and the keratinocyte sheet formation was analyzed, comparing various cell culture substrates (inactivated murine 3T3 fibroblasts, inactivated human fibroblasts, Collagen I or plain tissue culture plastic), as well as media containing serum or chemically defined animal‐free media.ResultsThe cell isolation step showed clear cell yield advantages when using porcine‐derived trypsin, compared to animal‐free alternatives. The keratinocyte sheets produced using animal‐free serum were similar to those produced using 3T3 feeder layer and FBS‐containing medium, particularly in mechanical integrity as all grafts were liftable. In addition, sheets grown on collagen in an animal‐free medium showed indications of advantages in homogeneity, speed, reduced variability, and differentiation status compared to the other growth conditions investigated. Most importantly, the procedure was compatible with the up‐scaling requirements of major burn wound treatments.ConclusionThis study demonstrated that animal‐free components could be used successfully to reduce the risk profile of large‐scale autologous keratinocyte sheet production, and thereby increase clinical accessibility.

Manufacturing autologous myoblast for regenerative medicine applications.

Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15-17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the donor age may impact the differentiation ability of myoblasts. The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases.

Improved self-renewal and differentiation potentials of mesenchymal Stem Cells by HPL-based xeno free Cell Rev™ mSC ExHQ 2.0

Background & Aim High-quality production and manufacturing of multipotent mesenchymal stem cells (MSCs) depend upon properties of culturing milieu of MSCs. Most importantly, the surrounding media must enhance self-renewal properties and retain differentiation potentials of MSCs namely the ability to turn into adipocytes, osteoblasts, chondrocytes, and others. Moreover, MSCs immunomodulatory advantages must be preserved in order for MSCs to be a therapeutic source of cell therapy. Common practices rely on the use of fetal bovine serum (FBS), dubbed standard media (STD), to support the expansion and of MSCs in vitro. Yet, FBS comes with intrinsic risks such as foreign proteins of bovine sources, viral or prion contamination and poses more possibilities of immune rejection, and reduces the immunomodulatory privileges of MSCs. To achieve a higher quality of biocompatibility and consistency for clinical applications, we formulated a critical recipe for MSCs cultured ex vivo to enhance their self-renewal and differentiation capacities. Methods, Results & Conclusion We sought to minimize the intrinsic risks from sera- and animal-free media while maximizing the critical features of MSCs. We, therefore, proposed a human platelet lysate-based, serum-free media, MSC ExHQ Cell Rev™ 2.0 media that can be used to proliferate MSCs while retaining their multipotent differentiation potentials. Here, we compare the properties of MSCs cultured in standard media (STD) versus in MSC ExHQ Cell Rev™ 2.0 media. MSCs grown in ExHQ Cell Rev™ 2.0 media show many improved qualities: 1) the effectiveness of colony-forming units (CFU) was 10-folds higher than STD, 2) the growth rate was 5-folds greater than STD. This was true up to passage 10, 3) differentiation to the 3 lineages were improved more than 3-folds, and 4) STD media causes overexpression of MSC negative marker, CD19 while ExHQ media does not. These data illustrate that MSC ExHQ Cell Rev™ 2.0 media is of notable choice for enhanced self-renewal and differentiation properties of MSCs.

InVivo Bioengineering of Fluorescent Conductive Protein-Dye Microfibers.

SummaryEngineering protein-based biomaterials is extremely challenging in bioelectronics, medicine, and materials science, as mechanical, electrical, and optical properties need to be merged to biocompatibility and resistance to biodegradation. An effective strategy is the engineering of physiological processes in situ, by addition of new properties to endogenous components. Here we show that a green fluorescent semiconducting thiophene dye, DTTO, promotes, in vivo, the biogenesis of fluorescent conductive protein microfibers via metabolic pathways. By challenging the simple freshwater polyp Hydra vulgaris with DTTO, we demonstrate the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. An integrated multilevel analysis including morphological, optical, spectroscopical, and electrical characterization shows electrical conductivity of biofibers, opening the door to new opportunities for augmenting electronic functionalities within living tissue, which may be exploited for the regulation of cell and animal physiology, or in pathological contexts to enhance bioelectrical signaling.

Selection of Osteoprogenitors from the Jaw Periosteum by a Specific Animal-Free Culture Medium

The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the in vitro culturing process efficiently. The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC-) in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs) show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1+ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1g L(-1) increased the maximal cell density by 70%, while a mixture of YE (1g L(-1)) and YP.A (4g L(-1)) increased IgG production by 180%. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20%. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

Cell-based medicinal products and the development of GMP-compliant processes and manufacturing.

When performing the GMP process development and scale up of cellular therapies, a critical review of the manufacturing process and all the materials and reagents involved in the production steps is the mandatory starting point to avoid potential issues related to the quality and safety of the product. The choice of the raw materials, plastics and all the additional equipment that comes into direct contact with the product must be performed always keeping in mind that cells, as drug products, cannot be terminally sterilized. The quality of the materials and reagents utilized is therefore directly related to the quality and degree of purity of the final product. Information about the available certification must be gathered for every component and, for critical materials, audits must be performed to the manufacturing sites to qualify the supplier. The protocols used for the cell expansion and processing (if necessary) must be designed trying to reduce at a minimum the dependence on growth factors and medium supplements. Each additional component that is added to the culture medium must be justified and its absence from the final product must be verified with validated methods. Residues that are not removed during the production steps must be accurately measured and limits must be set after performing a risk evaluation analysis, to ensure that these process-related impurities have no adverse effects on the patients. Supplements such as Fetal Bovine Serum (FBS) are permitted for the manufacturing of cellular therapies [1], as long as the serum is sourced from a TSE-free area. Anyway, the use of a medium containing FBS should be limited to the cases for which a valid alternative could not be found. However, continuous research and development is strongly advised in order to keep up to date with the latest advances in the field of medium formulations, in order to be ready to switch to an animal-free medium as soon as it is feasible. The reduction of growth factors and supplements is also effective in controlling the manufacturing costs of a cell therapy product. An evaluation of the economical aspects and market sustainability should be performed at an early stage to increase the chances for an industrial development of the cellular product. The manipulation steps performed during the manufacturing stage should be kept to a minimum, in order to reduce human intervention and decrease the risk of contamination. Media fill simulations must be performed in purposely stressed conditions to ensure that the process and the facility are able to support the production of a sterile product [2]. When manufacturing patient-specific therapies, extensive efforts should be directed towards the reduction in the variability of the starting material, that is usually a tissue sampled from the patient during hospitalization. Working with well-defined starting material allows for the set-up of a more robust process with comparable characteristics between batches dedicated to different patients. The specifications of the final product for parameters such as cell number, purity and potency must be wide enough to tolerate the normal biological variability of living organisms, but sufficiently narrowed down to generate comparable batches of drug. This uniformity is mandatory for the set up of clinical trials aiming at gathering a reliable analysis of the safety, tolerability and efficacy data obtained from treated patients, in order to * Correspondence: lromagnoli@aretaint.com Areta International s.r.l., Gerenzano (VA), 21040, Italy Romagnoli et al. BMC Proceedings 2011, 5(Suppl 8):O3 http://www.biomedcentral.com/1753-6561/5/S8/O3

Wound healing effects of gingival fibroblasts cultured in animal-free medium

The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal-free medium (HFDM1). We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5-2% was added to HFDM1. Wound healing cytokines, including transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL-6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Safety and Immunogenicity of CVD 1208S, a Live, Oral ΔguaBA Δsen Δset Shigella flexneri 2a Vaccine Grown on Animal-Free Media

A previous Phase 1 trial demonstrated that Shigella flexneri 2a deleted in guaBA, sen and set (strain CVD 1208) is well-tolerated and immunogenic after a single oral dose of 108 or 109 CFU. To facilitate further clinical development, the strain was reconstructed using animal-free media to conform to regulatory guidelines, and designated CVD1208S. Healthy inpatient volunteers were randomized (double-blind) to receive a single oral dose of either CVD 1208S (108 [n = 7] or 109 [n = 7] CFU) or placebo (n = 2). Both vaccine dosage levels were generally well-tolerated. Anti-lipopolysaccharide responses, measured as IgA antibody secreting cells, serum IgG, or fecal IgA levels, occurred in 7 (100%), 3 (43%) and 2 (29%) subjects, respectively, following inoculation with 109 CFU. Interferon gamma production in response to Shigella antigens was observed in 1 of 4 (25%) and 4 of 7 (57%) subjects, respectively, following inoculation with 108 and 109 CFU. We conclude that CVD 1208S retains a favorable safety and immunogenicity profile after reconstruction on animal-free media, comparable to that seen with CVD 1208, which was constructed on media containing animal products, and shows promise as a live, oral Shigella vaccine.

Fabrication of Cultured Oral Gingiva by Tissue Engineering Techniques Without Materials of Animal Origin

Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture.

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3x10(6) viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.