Serum-free Media Formulations Research Articles

Environmental life cycle assessment of recombinant growth factor production for cultivated meat applications

Growth factors are critical components of current serum-supplemented and serum-free media formulations for cultivated meat production. However, growth factors have been excluded, estimated using proxies, or modeled using proprietary data in existing environmental assessments of cultivated meat products. Cell culture media has been identified as a hotspot in such studies, therefore it is important to accurately quantify the environmental impacts of growth factor supplementation. To address this gap, this study applied life cycle assessment (LCA) methodology to comparatively assess the environmental impacts of recombinant growth factor production for cultivated meat applications. Life cycle inventories were developed for four recombinant growth factors (IGF-1, FGF, TGF-β, and PDGF) produced using a novel bench-scale process. The functional unit was selected as 1 mg of produced growth factor. The results indicate that recombinant growth factors can have significant environmental impacts within cultivated meat systems, despite being used in very small quantities. For example, the global warming potential of production of 1 mg of IGF-1, FGF, TGF-β, and PDGF was estimated to be 0.1, 0.04, 0.2 and 0.2 kg CO2 eq, respectively. Future research should explore the sustainability of producing food-grade growth factors at scale to meet the needs of the expanding cultivated meat industry or identifying alternatives to growth factors that have lower impacts on the environment.

Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates

The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is the high-cost of recombinant albumin which is necessary in many serum-free media formulations, including a recently developed serum-free medium for bovine satellite cell (BSC) culture termed Beefy-9. Here we alter Beefy-9 by replacing recombinant albumin with rapeseed protein isolate (RPI), a bulk-protein solution obtained from agricultural waste through alkali extraction (pH 12.5), isoelectric protein precipitation (pH 4.5), dissolution of physiologically soluble proteins (pH 7.2), and concentration of proteins through 3 kDa ultrafiltration. This new medium, termed Beefy-R, was then used to culture BSCs over four passages, during which cells grew with an average doubling time of 26.6 h, showing improved growth compared with Beefy-9. In Beefy-R, BSCs maintained cell phenotype and myogenicity. Together, these results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production.

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.

Markedly enhanced Mesenchymal Stromal Cell growth in xeno- and serum-free media formulations supplemented with fibrinogen-depleted human platelet lysate

Background & Aim With increasing demand for Mesenchymal Stromal Cell (MSC) therapy, there is a need for reproducible chemically defined xeno- and serum-free media for growth of hMSCs to minimize process related variability. In the present study, we compare 4 commercially available xeno- and serum-free media (SFM 1-4) from different manufacturers with traditional Dulbeco's Modified Eagle Media (DMEM) containing fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (hPL). Additionally, SFM from one supplier was further tested in combination with or without hPL. Critical hMSC attributes including cell yield, doubling times (dt), population doublings (pd), morphology, identity through flow cytometry and in vitro potency through the induction of indoleamine 2,3-dioxygenase (IDO) by interferon-gamma (IFN-γ) were recorded. Methods, Results & Conclusion MSC yields were significantly higher in SFM (SFM1: 25600, SFM2: 16000, SFM3: 38267 and SFM4: 21600 cells/cm2) when compared to DMEM containing FBS (2000 cells/cm2) or hPL (4133 cell/cm2). Additionally, cells cultured in all SFMs had spindle shaped morphology and appeared smaller in size (medium diameter in SFM: 17.0 ± 1.3 µm versus hPL: 20.4 µm). Cells grown in SFM conditions had significantly lower doubling times (SFM1: 25.4h, SFM2: 28.8h, SFM3: 27.4h and SFM4: 26.5h vs. FBS: 72.0h and hPL: 47.3h) and higher population doublings (SFM1: 5.7, SFM2: 5.0, SFM3: 6.3 and SFM4: 5.4 versus FBS: 2.0 and hPL: 3.1). This effect was heightened in SFM1 basal media with hPL (yield: 54440 cells/cm2; dt: 20.8h and pd: 6.8) and even further with a combination of SFM1 complete media with fibrinogen depleted hPL (92667 cells/cm2; dt: 18.4h, pd: 7.5). There were no differences in terms of cell identity in different media formulations. All cells were positive (>95% expression) for CD73, CD90, CD105 and negative (<5% expression) for CD14, CD19, CD34, CD45, HLA-DR. Cells cultured in SFM were equally potent as those cultured in hPL or FBS as indicated by enhanced IDO expression when treated with IFN-γ (ΔΔCt; SFM: 15.7 vs FBS: 15.6 and hPL: 16.1). Interestingly, improved IDO expression was observed in cells cultured in SFM1 supplemented with hPL (ΔΔCt: 17.6). In conclusion, hMSCs have markedly faster growth but no impact on identity or in vitro potency when cultured in specific SFM formulations. These markedly enhanced rates of proliferation may provide manufacturing advantages yet the ultimate impact on cell phenotype and product safety is yet to be determined. With increasing demand for Mesenchymal Stromal Cell (MSC) therapy, there is a need for reproducible chemically defined xeno- and serum-free media for growth of hMSCs to minimize process related variability. In the present study, we compare 4 commercially available xeno- and serum-free media (SFM 1-4) from different manufacturers with traditional Dulbeco's Modified Eagle Media (DMEM) containing fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (hPL). Additionally, SFM from one supplier was further tested in combination with or without hPL. Critical hMSC attributes including cell yield, doubling times (dt), population doublings (pd), morphology, identity through flow cytometry and in vitro potency through the induction of indoleamine 2,3-dioxygenase (IDO) by interferon-gamma (IFN-γ) were recorded. MSC yields were significantly higher in SFM (SFM1: 25600, SFM2: 16000, SFM3: 38267 and SFM4: 21600 cells/cm2) when compared to DMEM containing FBS (2000 cells/cm2) or hPL (4133 cell/cm2). Additionally, cells cultured in all SFMs had spindle shaped morphology and appeared smaller in size (medium diameter in SFM: 17.0 ± 1.3 µm versus hPL: 20.4 µm). Cells grown in SFM conditions had significantly lower doubling times (SFM1: 25.4h, SFM2: 28.8h, SFM3: 27.4h and SFM4: 26.5h vs. FBS: 72.0h and hPL: 47.3h) and higher population doublings (SFM1: 5.7, SFM2: 5.0, SFM3: 6.3 and SFM4: 5.4 versus FBS: 2.0 and hPL: 3.1). This effect was heightened in SFM1 basal media with hPL (yield: 54440 cells/cm2; dt: 20.8h and pd: 6.8) and even further with a combination of SFM1 complete media with fibrinogen depleted hPL (92667 cells/cm2; dt: 18.4h, pd: 7.5).

Evaluation of serum-free media formulations in feeder cell–stimulated expansion of natural killer cells

BackgroundOptimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum–containing medium for their ability to support NK cell expansion and function. MethodsHuman peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. ResultsTexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DiscussionThe AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.

Cell-Based Fish: A Novel Approach to Seafood Production and an Opportunity for Cellular Agriculture

Cellular agriculture is defined as the production of agricultural products from cell cultures rather than from whole plants or animals. With growing interest in cellular agriculture as a means to address the public health, environmental, and animal welfare challenges of animal agriculture, the concept of producing seafood from fish cell- and tissue-cultures is emerging as a means to address similar challenges with industrial aquaculture systems and marine capture. Cell-based seafood - as opposed to animal-based seafood - can combine developments in biomedical engineering with modern aquaculture techniques. Biomedical engineering developments such as closed-system bioreactor production of land animal cells create a basis for large scale production of marine animal cells. Aquaculture techniques such as genetic modification and closed system aquaculture have achieved marked gains in production that can pave the way for innovations in cell-based seafood production. Here, we present the current state of innovation relevant to the development of cell-based seafood across multiple species as well as specific opportunities and challenges that exist for advancing this science. The authors find that the physiological properties of fish cell- and tissue- culture may be uniquely suited to cultivation in vitro. These physiological properties, including hypoxia tolerance, high buffering capacity, and low-temperature growth conditions, make marine cell culture an attractive opportunity for scale production of cell-based seafood; perhaps even more so than mammalian and avian cell cultures for cell-based meats. This, coupled with the unique capabilities of crustacean tissue-friendly scaffolding such as chitosan, a common seafood waste product and mushroom derivative, presents great promise for cell-based seafood production via bioreactor cultivation. To become fully realized, cell-based seafood research will require more understanding of fish muscle culture and cultivation; more investigation into serum-free media formulations optimized for fish cell culture; and bioreactor designs tuned to the needs of fish cells for large scale production.

Evaluation of serum-free media formulations in feeder cell expansion of natural killer cells

Background & Aim Natural Killer (NK) cells are rapidly evolving as a cellular therapy due to their innate ability to kill a wide range of cancerous cells without prior sensitization and broad safety profile without concerns for causing graft-versus host disease. However, the clinical grade production of NK cell products has historically required the addition of fetal bovine or human serum, which have safety, ethical, and regulatory concerns. Serum-free media have been established for T cell activation and expansion, but little is known about serum-free media in manufacturing NK cells. Here, we sought to assess a broad range of serum-free media in supporting NK cell expansion with K562-based feeder cells for use in adoptive immunotherapy. Methods, Results & Conclusion Cryopreserved expanded human peripheral blood NK cells (ePBNK) were re-stimulated at 1:1 ratio with irradiated K562 expressing mbIL21 and 4-1BBL (CSTX002) and propagated for 7 days in 6 different clinical-grade serum-free media. The three serum-free media that yielded the highest cell numbers (TexMACS, ABS011, and AIM-V/ICSR) were further compared for support of 2-week NK cell expansion starting from freshly-isolated PBNK, and the resulting ePBNK were assessed for phenotype, cytotoxicity, degranulation, and cytokine production. NK cells did not have acceptable expansion kinetics with OpTmizer, SCGM, or StemXVivo. Surprisingly, NK cells had significantly improved expansion with AIM-V/ICSR (5448-fold) compared to RPMI + FBS control (2621-fold). Additionally, the AIM-V/ICSR expanded NK cells showed increased anti-tumor cytotoxicity and TNFa secretion, and had increased expression of several NK cell activating receptors and effector molecules such as DNAM-1, NKG2D, NKp30, FasL, granzyme B, and perforin. These results indicate that the serum-free AIM-V/ICSR expansion medium can support ex vivo expansion of NK cells with high effector function and phenotype compared to the standard RPMI + FBS medium and is an effective alternative for use in adoptive NK cell therapy.

Developing scalable cultivation systems of hepatic spheroids for drug metabolism via genomic and functional analyses.

Spheroids, a widely used three-dimensional (3D) culture model, are standard in hepatocyte culture as they preserve long-term hepatocyte functionality and enhance survivability. In this study, we investigated the effects of three operation modes in 3D culture - static, orbital shaking, and under vertical bidirectional flow using spheroid forming units (SFUs) on hepatic differentiation and drug metabolism to propose the best for mass production of functionally enhanced spheroids. Spheroids in SFUs exhibited increased hepatic gene expression, albumin secretion, and cytochrome P450 3A4 (CYP3A4) activity during the differentiation period (12 days). SFUs advantages include facilitated mass production and a relatively earlier peak of CYP3A4 activity. However, CYP3A4 activity was not well maintained under dimethyl sulfoxide (DMSO)-free conditions (13-18 days), dramatically reducing drug metabolism capability. Continued shear stimulation without differentiation stimuli in assay conditions markedly attenuated CYP3A4 activity, which was less severe in static conditions. In this condition, SFU spheroids exhibited dedifferentiation characteristics, such as increased proliferation and Notch signaling genes. We found that the dedifferentiation could be overcome by using the serum-free medium formulation. Therefore, we suggest that SFUs represent the best option for the mass production of functionally improved spheroids and so the serum-free conditions should be maintained during drug metabolism analysis.

A serum-free medium formulation efficiently supports isolation and propagation of canine adipose-derived mesenchymal stem/stromal cells.

Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato’s approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.

On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm

Substitution of serum and other clinically incompatible reagents is requisite for controlling product quality in a therapeutic cell manufacturing process. However, substitution with chemically defined compounds creates a complex, large-scale optimization problem due to the large number of possible factors and dose levels, making conventional process optimization methods ineffective. We present a framework for high-dimensional optimization of serum-free formulations for the expansion of human hematopoietic cells. Our model-free approach utilizes evolutionary computing principles to drive an experiment-based feedback control platform. We validate this method by optimizing serum-free formulations for first, TF-1 cells and second, primary T-cells. For each cell type, we successfully identify a set of serum-free formulations that support cell expansions similar to the serum-containing conditions commonly used to culture these cells, by experimentally testing less than 1 × 10−5 % of the total search space. We also demonstrate how this iterative search process can provide insights into factor interactions that contribute to supporting cell expansion.

Fetal bovine serum (FBS): Past – present – future

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

A Novel Protocol for Directed Differentiation of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes.

Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient, novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases.

Serum-free media formulations are cell line–specific and require optimization for microcarrier culture

Background aimsMesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. MethodsWe cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. ResultsWe show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. ConclusionsCurrent serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line.

Abstract 5360: Towards identification of cancer stem cells in esophageal adenocarcinoma

Abstract Introduction: Cancer stem cells (CSC) are characterized by stem cell properties such as self-renewal potential and multilineage differentiation capacity. CSC's are thought to be the main drivers of tumor formation and progression and the main cause of therapy resistance and metastatic spread. In contrast to other cancer types such as glioblastoma and colon cancer, attempts thus far have not resulted in the identification of a proven CSC population in esophageal cancer. Therefore in this study we aim to identify and characterize CSC's in esophageal adenocarcinoma (EAC). Methods and Results: To address this goal, we attempted to culture CSC's from primary untreated EAC biopsy material using serum-free medium formulations that, however, have not been successful thus far. As an alternative model we employed two EAC cell lines, OE19 and OE33, which we were able to maintain as spheroids in serum-free Neural Basal Medium thought to enrich for stem cell growth. Dissociation of spheres and limiting dilution assays showed enhanced spheroid forming potential in OE19 but not in OE33 when compared to cells obtained from normal 10% FCS monolayer cultured counterparts. Next, the expression of several known CSC markers was determined by FACS analysis to determine self-renewal potential of sorted fractions in subsequent limited dilution assays. Side population, ALDH1, CD44/CD24 fractions were tested of which only CD44+ /CD24+ cells showed enhanced capacity to form spheroids. Tumor growths of these populations are currently being evaluated on immune-compromised mice. Other stem cell markers such as OCT4 and SOX2 were enhanced in OE19 spheroids compared to 10% FCS cultured cells. However, in OE33 spheroids expression of these markers were unchanged or downregulated. Since EAC derives from dysplastic epithelia with intestinal characteristics in Barrett's esophagus, we examined the status of the Wnt pathway known to be involved in maintenance of intestinal/ colon (cancer) stem cells. In the EAC cells β-catenin expression was present that could be enhanced by LiCl. Reporter assays to determine Wnt- activity are in progress. Finally, the sensitivity of spheroid versus monolayer cultured OE33 cells to 5-FU, cisplatin and paclitaxel was evaluated. Sensitivity did not differ much, although monolayer cultured OE33 cells were more resistant for 5-FU. Conclusion: Given the differences in the two spheroid models regarding spheroid forming potential, CSC-marker expression and chemosensitivity, caution is warranted when using esophageal adenocarcinoma cell line derived spheroids as model for CSC's. However, CD44 and CD24 might have a potential as CSC-markers in EAC, which is currently further corroborated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5360. doi:1538-7445.AM2012-5360

Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin®bioreactors

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells ( Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8–9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133 +CD24 −/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca 2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers

The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca 2+ currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinson’s. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback.

222 Ofatumumab binds to a membrane-proximal epitope which comprises amino acids in the small and large loops of CD20

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005, Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8–9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133+CD24−/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Serum-free cell culture: the serum-free media interactive online database

Fetal bovine serum (FBS) is a ubiquitously used essential supplement in cell culture media. However, there are serious scientific and ethical concerns about the use of FBS regarding its harvest and production. During the last three decades, FBS could be substituted by other supplements or by the use of defined chemical components in serum-free cell culture. A number of serum-free medium formulations have been described for mammalian and insect cell lines as well as for primary cultures. However, the switch to serum-free media still demands a time-consuming literature survey and a manufacturer search for appropriate medium formulations, respectively. Here we present the second collection of commercially available serum-free media in an updated, freely accessible interactive online database. Searches for serum-free media and continuous cell lines already adapted to serum-free culture can be performed according to various criteria. These include the degree of chemical definition, e.g. serum-free (SF), animal-derived component free (ADCF) or chemically defined (CD), and the type of medium, e.g. basal media, medium supplements, or full replacement media. In order to specify the cell lines that are adapted to serum-free media, search terms like species, organ, tissue, cell type and disease can be used. All commercially available serum-free media and adapted cell lines currently available from major distributors (e.g. ATCC, ECACC and DMSZ) are included in the database. Despite an extensive search for serum-free media and adapted cell lines, detailed information from certain companies and suppliers is still lacking and is specifically highlighted. It is intended to create a platform for the interactive exchange of information and experience by experts in the field in order to continuously improve and extend the serum-free online database. The database is accessible at http://www.goodcellculture.com/