Chitosan/Cellulose-Based Porous Nanofilm Delivering C-Phycocyanin: A Novel Platform for the Production of Cost-Effective Cultured Meat.

Cultured meat is artificial meat produced via the mass culture of cells without slaughtering livestock. In the production process of cultured meat, the mass proliferation for preparing abundant cells is a strenuous and time-consuming procedure requiring expensive and excess serum. Herein, C-phycocyanin (C-PC) extracted from blue algae was selected as a substitute for animal-derived serum and a polysaccharide film-based platform was developed to effectively deliver C-PC to myoblast while reducing the cost of cell medium. The polysaccharide platform has a sophisticated structure in which an agarose layer is capped on a porous multilayer film formed by molecular reassembly between chitosan and carboxymethylcellulose (CMC). The porous multilayer film provides an inner structure in which C-PC can be incorporated, and the agarose layer protects and stabilizes the C-PC. The completed platform was easily applied to a cell culture plate to efficiently release C-PC, thereby improving myoblast proliferation in a serum-reduced environment during long-term culture. We developed a cell sheet-based meat model using this polysaccharide platform to evaluate the improved cost-efficiency by the platform method in the mass proliferation of cells. This strategy and innovative technology can simplify the production system and secure price competitiveness to commercialize cultured meat.

Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

Homogenization of initial cell distribution by secondary flow of medium improves cell culture efficiency.

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.

Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays.

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.

Cost of decentralized CAR T-cell production in an academic nonprofit setting.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy with high acquisition costs, and it has raised concerns about affordability and sustainability in many countries. Furthermore, the current centralized production paradigm for the T cells is less than satisfactory. Therefore, several countries are exploring alternative T-cell production modes. Our study is based on the T-cell production experience in a nonprofit setting in Germany. We first identified the work steps and main activities in the production process. Then we determined the fixed costs and variable costs. Main cost components included personnel and technician salaries, expenditure on equipment, a clean room, as well as production materials. All costs were calculated in 2018 euros and converted into U.S. dollars. For a clean room with one machine for closed and automated manufacturing installed, annual fixed costs summed up to approximately €438 098 ($584 131). The variable cost per production was roughly €34 798 ($46 397). At the maximum capacity of one machine, total cost per product would be close to €60 000 ($78 849). As shown in the scenario analysis, if three machines were to be installed in the clean room, per production cost could be as low as €45 000 (roughly $59905). If a cheaper alternative to lentivirus was used, per production total cost could be further reduced to approximately €33 000 (roughly $44309). Decentralized T-cell production might be a less costly and more efficient alternative to the current centralized production mode that requires a high acquisition cost.

Cost-effective culture of human induced pluripotent stem cells using UV/ozone-modified culture plastics with reduction of cell-adhesive matrix coating.

Human induced pluripotent stem cells (hiPSCs) are considered to be one of the most promising cell resources for regenerative medicine. HiPSCs usually maintain their pluripotency when they are cultured on feeder cell layers or are attached to a cell-adhesive extracellular matrix. In this study, we developed a culture system based on UV/ozone modification for conventional cell culture plastics to generate a suitable surface condition for hiPSCs. Time of flight secondary ion mass spectrometry (ToF-SIMS) was carried out to elucidate the relationship between hiPSC adhesion and UV/ozone irradiation-induced changes to surface chemistry of cell culture plastics. Cell culture plastics with modified surfaces enabled growth of a feeder-free hiPSC culture with markedly reduced cell-adhesive matrix coating. Our cell culture system using UV/ozone-modified cell culture plastics may produce clinically relevant hiPSCs at low costs, and can be easily scaled up in culture systems to produce a large number of hiPSCs.

Decellularized matrices for tumor cell modeling.

Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L-1 ·day-1 , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.

Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture.

Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor ß3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.

Rapid generation of functional hepatocyte-like cells from human minor salivary gland-derived stem cells.

Liver failure is one of the major risk factors for death worldwide, and the only effective liver transplantation is currently very limited. Adult stem cells can be induced into hepatocytes in vitro and implanted into the body to repair damaged liver. However, most of the induction time in vitro is relatively long, which is not suitable for practical application. Therefore, search for new seed cells that can rapidly differentiate into functional hepatocytes is crucial for the clinical application of cell transplantation therapy. In this study, we explored a three-step protocol to rapidly induce human minor salivary gland mesenchymal stem cells (hMSG-MSCs) into hepatocytes in vitro, and finally obtained hepatocyte-like cells within 6 days. After a series of relevant detection from gene, protein and functional levels, we confirmed that the finally induced cells were mature hepatocyte-like cells with certain hepatocyte functions to some extent. Besides, we injected the preliminary induced cells into mice with acute liver injury, showing a good repair effect on the damaged liver. All these results indicate that the hMSG-MSCs have potential to be a kind of seed cells for rapid hepatic differentiation.

A cost-effective three-dimensional culture platform functionally mimics the adipose tissue microenvironment surrounding the kidney.

There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.

Low-cost cultivation and sporulation of alkaliphilic Bacillus sp. strain AK13 for self-healing concrete.

The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 108 and 1.25 ± 0.12 × 108 CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 109 CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 106 CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.

Fidelity of a PDX-CR model for bladder cancer.

Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

A rapid cell-based assay for determining poloxamer quality in CHO suspension cell culture.

Poloxamers are water-soluble polymers that are widely used in cell culture bioprocessing to protect cells against shearing forces. Use of poor-quality poloxamers may lead to a drastic reduction in cell growth, viabilities and productivities in cell culture-based manufacturing. In order to evaluate poloxamer quality and promote more consistent performance, a rapid cell membrane adhesion to hydrocarbon assay was developed based on the adhesive properties of cell membranes to selective hydrocarbons. The assay can identify a poor-performing poloxamer characterized by significant drop in viable cell density and percent viability. The assay was verified across multiple good and bad poloxamer lots, and the results were in agreement with established cell growth and high-performance liquid chromatography assays.

Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms.

Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.

Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical-Wheel Bioreactor System: An Experimental and Economic Approach.

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering applications and cell-based therapies. Large cell doses (>1 × 106 cells kg-1 ) and Good Manufacturing Practices (GMP)-compliant processes are however required for clinical purposes. Here, a serum- and xenogeneic-free (S/XF) microcarrier-based culture system is established for the expansion of human umbilical cord matrix (UCM)- and adipose tissue (AT)-derived MSC using the Vertical-Wheel system (PBS-0.1 MAG; PBS Biotech). UCM and AT MSC are expanded to maximum cell densities of 5.3 ± 0.4 × 105 cell mL-1 (n = 3) and 3.6 ± 0.7 × 105 cell mL-1 (n = 3), respectively, after 7 days of culture, while maintaining their identity, according to standard criteria. An economic evaluation of the process transfer from T-flasks to PBS-0.1 MAG shows a reduction in the costs associated with the production of a dose for an average 70 kg adult patient (i.e., 70 million cells). Costs decrease from $17.0 K to $11.1 K for UCM MSC and from $21.5 K to $11.1 K for AT MSC, proving that the transition to Vertical-Wheel reactors provides a cost-effective alternative for MSC expansion. The present work reports the establishment of a scalable and cost-effective culture platform for the manufacturing of UCM and AT MSC in a S/XF microcarrier-based system.

Organoid culture media formulated with growth factors of defined cellular activity.

The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.

Modeling biological and economic uncertainty on cell therapy manufacturing: the choice of culture media supplementation.

AIM: To evaluate the cost-effectiveness of autologous cell therapy manufacturing in xeno-free conditions. MATERIALS & METHODS: Published data on the isolation and expansion of mesenchymal stem/stromal cells introduced donor, multipassage and culture media variability on cell yields and process times on adherent culture flasks to drive cost simulation of a scale-out campaign of 1000 doses of 75 million cells each in a 400 square meter Good Manufacturing Practices facility. RESULTS & CONCLUSION: Passage numbers in the expansion step are strongly associated with isolation cell yield and drive cost increases per donor of $1970 and 2802 for fetal bovine serum and human platelet lysate. Human platelet lysate decreases passage numbers and process costs in 94.5 and 97% of donors through lower facility and labor costs. Cost savings are maintained with full equipment depreciation and higher numbers of cells per dose, highlighting the number of cells per passage step as the key cost driver.