Cell Culture Production Research Articles

Methods for identifying precipitates and improving stability of chemically defined highly concentrated cell culture media.

Currently, within the biopharmaceutical industry, media development is a key area of development as the ratios and concentrations of media components such as amino acids, metals, vitamins, sugars, salts, and buffering agents play arguably the largest role in cellular productivity and product quality. However, optimizing media for these targets often conflicts with solubility limitations and slow-rate chemical reactions that result in precipitation formation. Here we present methods such as inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence (XRF), colorimetry, and turbidity to identify multiple likely components of a complex precipitate that was observed in preparations of a custom nutrient feed medium across all storage conditions evaluated. Using these analytical methods, as well as adjustments to the formulation pH, increasing the pyruvate concentration, and removing sodium bicarbonate, we were able to extend the media shelf life from approximately 10 days to over 28 days. Alternatively, copper, selenium, and magnesium sources were removed from the media and no precipitation was observed until 32 days after prep, pointing to key metals as the probable root cause of precipitation. By analytically quantifying the precipitate using the methods above, instead of visual inspection, which is the current industry standard for media precipitation observation, we were better able to compare conditions to one another and relate them to the onset of precipitation. Cell culture performance and product quality remained comparable to the historical process despite the media formulation changes.

Composition and Bioactivity of a Placental Tissue Particulate (PTP-001) Indicate Greater Potential than Platelet-Rich Plasma for the Treatment of Osteoarthritis.

This study was conducted to compare therapeutically relevant properties of platelet-rich plasma (PRP), a commonly used autologous intra-articular treatment for osteoarthritis (OA), with those of a novel placental tissue particulate, PTP-001, which is in development as a regulated biologic treatment for knee OA. Quantitative immunoassays were performed to determine the content of key growth/regulatory biofactors in PTP-001, and in leukocyte-rich (LR)-PRP or leukocyte-poor (LP)-PRP. An anti-inflammatory bioassay was used to evaluate the effects of each treatment on pro-inflammatory cytokine (tumor necrosis factor (TNF)-α) production in a macrophage cell culture system. Gene expression experiments were conducted using a co-culture system of human synoviocytes (pre-stimulated with interleukin (IL)-1β) and articular chondrocytes, with quantitative polymerase chain reaction analyses of the separate cellular compartments. The concentrations of several biofactors (e.g., basic fibroblast growth factor, tissue inhibitor of metalloproteases-3, interleukin-1 receptor antagonist) representative of diverse disease-relevant mechanisms of action were significantly higher for PTP-001 relative to LR-PRP or LP-PRP. PTP-001 and PRP preparations were able to reduce TNF-α production in macrophage cell cultures; however, greater variability was observed for PRP in comparison with PTP-001. In the chondrocyte/synoviocyte co-culture experiments, PTP-001 and LR-PRP (but not LP-PRP) significantly reduced chondrocyte MMP13 expression in cultures containing IL-1-pretreated synoviocytes. In addition, ADAMTS5 expression was reduced in the chondrocyte compartment following treatment with PTP-001 relative to PRP. These findings support evidence of a potent, multifactorial mechanism of action for a consistently manufactured biologic (PTP-001), which may be of greater therapeutic benefit in comparison with more heterogeneous preparations of PRP which may be generated at the time of treatment.

Improving fed-batch culture efficiency of Rhodiola sachalinensis cells and optimizing flash extraction process of polysaccharides from the cultured cells by BBD–RSM

Fed-batch culture of plant cells can enhance biomass and bioactive compound accumulation compared with batch culture, in which the initial culture medium is one of the controllable factors in the fed-batch culture system. Rhodiola sachalinensis is a valuable medicinal herb and its cell culture is an alternative approach to obtaining plant material. R. sachalinensis cells has been previously cultured in a fed-batch culture system and the initial culture medium has been found to critically affect production yield. Therefore, this study investigated the effect of nitrogen concentration, ammonia and nitrate ratio (NH4+/NO3-), and phosphorus and calcium concentrations in initial culture medium on cell biomass, as well as salidroside and polysaccharide accumulation to improve the fed-batch culture efficiency. Then, the cultured cells were extracted by flash extraction and the extraction conditions were optimized by Box-Behnken Design Response Surface Method (BBD–RSM). Finally, the antibacterial and antibiofilm effects of the extract were examined to further utilize R. sachalinensis cell cultures. The results showed that the fed-batch culture efficiency was obviously improved when the initial culture medium was modified as 30 mM of nitrogen with 5/25 of NH4+/NO3-, 0.94 mM of phosphorus, and 2.3 mM of calcium, at which 277.3 mg/L of salidroside and 4.5 g/L of polysaccharides were obtained In flash extraction, polysaccharide yield was as the response value to optimize extraction process by controlling extraction time, extraction temperature, and liquid–solid ratio in the BBD–RSM experiments. The optimal flash extraction conditions were 55.7 s of extraction time, 56.5 °C of extraction temperature, and 37.0 mL/g of liquid–solid ratio, which the polysaccharide yield reached 12.76% under the optimized conditions. In antibacterial experiment, the cell extract (RCE) inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Salmonella choleraesuis; the biofilm of B. subtilis and S. choleraesuis was scavenged in the groups of 8 mg/mL and 16 mg/mL RCE. The findings provided a basis for further industrial production of cell cultures and a theoretical reference in using R. sachalinensis cells in the production of antibacterial-related products.

Genetic approaches in improving biotechnological production of taxanes: An update.

Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agents in the treatment of various types of cancer. Due to the scarcity of PTX in nature, its production in cell cultures and plant organs is a major challenge for plant biotechnology. Although significant advances have been made in this field through the development of metabolic engineering and synthetic biology techniques, production levels remain insufficient to meet the current market demand for these powerful anticancer drugs. A key stumbling block is the difficulty of genetically transforming the gymnosperm Taxus spp. This review focuses on the progress made in improving taxane production through genetic engineering techniques. These include the overexpression of limiting genes in the taxane biosynthetic pathway and transcription factors involved in its regulation in Taxus spp. cell cultures and transformed roots, as well as the development and optimization of transformation techniques. Attempts to produce taxanes in heterologous organisms such as bacteria and yeasts are also described. Although promising results have been reported, the transfer of the entire PTX metabolic route has not been possible to date, and taxane biosynthesis is still restricted to Taxus cells and some endophytic fungi. The development of a synthetic organism other than Taxus cells capable of biotechnologically producing PTX will probably have to wait until the complete elucidation of its metabolic pathway.

Effect of new azoloazine derivatives with potential antitumor activity on energy metabolism in MCF-7 and Vero cell cultures

Introduction. The work presents the results of studying the effects of three new azoloazine derivatives on oxidative glucose metabolism in order to select substances with the most acceptable characteristics for further preclinical study as potential antitumor agents, including for breast cancer chemotherapy.Aim. The aim of the work is to identify the metabolic properties of new azoloazine derivatives in terms of their effect on glucose metabolism using a culture of MCF-7 tumor cells and Vero non-tumor cells.Material and Methods. The testing on cell cultures was the main method used in the work, and all tested compounds were applied in final concentrations from 2.5 μmol/L. The comparison drug was epirubicin in the same concentration. The biochemical techniques included the determination of lactate production using commercial Olvex Diagnosticum kits and the determination of oxygen consumption by cells using the Seahorse XFe24 Analyzer for cellular metabolism. The results were processed statistically.Results. Lactate production in MCF-7 and Vero cell cultures decreased by more than half in the presence of 3-Cyclohexyl4-oxoimidazo[5,1-d]-[1,2,3,5]tetrazine-8-N-piperidinyl-carboxamide, and oxygen consumption decreased by 19-40%, which was the maximum effect among the studied azoloazine derivatives. Diethyl ether of 4-aminoimidazo[5,1-c][1,2,4]triazine-3,8dicarboxylic acid and 4-Amino-8-ethoxycarbonyl-imidazo[5,1-c][1,2,4]triazine-3-N-(p-toluyl)carboxamide were similar in their metabolic effects to the comparison drug epirubicin. They reduced lactate production in MCF-7 and Vero cell culture by a third and by 21–22%, respectively. Oxygen consumption in MCF-7 cell culture decreased by 14–17%, in Vero cell culture it decreased by 18–24%.Conclusion. The data obtained allow us to consider the (3-Cyclohexyl-4-oxoimidazo[5,1-d]-[1,2,3,5]tetrazine-8-N-piperidinylcarboxamide as the leader among new azoloazine derivatives and recommend it for further preclinical study as a potential antitumor agent.

Antioxidant Green Factories: Toward Sustainable Production of Vitamin E in Plant In Vitro Cultures.

Vitamin E is a dietary supplement synthesized only by photosynthetic organisms and, hence, is an essential vitamin for human well-being. Because of the ever-increasing demand for natural vitamin E and limitations in existing synthesis modes, attempts to improve its yield using plant in vitro cultures have gained traction in recent years. With inflating industrial production costs, integrative approaches to conventional bioprocess optimization is the need of the hour for multifold vitamin E productivity enhancement. In this review, we briefly discuss the structure, isomers, and important metabolic routes of biosynthesis for vitamin E in plants. We then emphasize its vital role in human health and its industrial applications and highlight the market demand and supply. We illustrate the advantages of in vitro plant cell/tissue culture cultivation as an alternative to current commercial production platforms for natural vitamin E. We touch upon the conventional vitamin E metabolic pathway engineering strategies, such as single/multigene overexpression and chloroplast engineering. We highlight the recent progress in plant systems biology to rationally identify metabolic bottlenecks and knockout targets in the vitamin E biosynthetic pathway. We then discuss bioprocess optimization strategies for sustainable vitamin E production, including media/process optimization, precursor/elicitor addition, and scale-up to bioreactors. We culminate the review with a short discussion on kinetic modeling to predict vitamin E production in plant cell cultures and suggestions on sustainable green extraction methods of vitamin E for reduced environmental impact. This review will be of interest to a wider research fraternity, including those from industry and academia working in the field of plant cell biology, plant biotechnology, and bioprocess engineering for phytochemical enhancement.

Role of geneticin in isolation and culturing of skin melanocytes and melanoma cells

Abstract Introduction Development of an effective, suitable, and reliable method for both the isolation and culturing of melanocytes is crucial for studies on pathomechanisms of skin diseases originating from melanocyte disorders. In this study, we have investigated the influence of geneticin (G418), a substance used for melanocyte selection, in the view of the frequency of presence of cells such as keratinocytes and fibroblasts, widely known as contaminators of melanocyte-originating cell cultures. Materials and Methods Study was conducted on primary, freshly isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells as well as on commercially available melanoma cell lines MeWo, G-361, and A375. Cells were cultured in different culture media supplemented with various concentrations of geneticin ranging from 0.05 to 1 mg/mL. Cell viability, proliferation rate and detection of apoptotic/necrotic cells was assessed. Results Choice of culture media supplemented with various concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) strongly affect viability of melanocytes, fibroblasts, and keratinocytes. Selective culture media without FBS facilitate the process of melanocytes and melanoma pure cell culture, yet without geneticin supplementation are insufficient for complete eradication of fibroblast contamination from cell culture. Conclusions In this study we provide, for the first time, the dose-response action of keratinocytes and fibroblasts upon geneticin stimulation in different culture media and show that a low concentration (0.05 mg/mL) of geneticin added to the selective culture media may be safely implemented to facilitate the production of melanocyte and melanoma cell cultures that are free from frequent cell contaminants.

The peptide fraction of Bothrops jararaca snake venom induces toxicological effects on the male reproductive system after local envenomation in mice

Bothrops envenomation is complex and provokes prominent local tissue damage and systemic disturbances, but little is known about their effects on the male reproductive system. After intratesticular injection, the bioactive peptide fraction (Bj-PF) obtained from Bothrops jararaca snake venom changes the structure of different stages of the seminiferous epithelium cycle in adult mice. For the first time, we investigated whether local envenomation of Bj-PF induces toxicological effects on the male reproductive system, particularly on the seminiferous epithelium and Sertoli cells. Male adult mice were treated with 0.24 mg.kg−1 by intramuscular (i.m.) injection for 24 h. The testes samples were collected for morphological and morphometric evaluation. The toxicological effects of Bj-PF were also analyzed on mitochondrial metabolism and nitrite (NO2) production in 15P-1 Sertoli cell culture. Bj-PF changed the structure and function of the seminiferous epithelium, particularly the disruption of the epithelium and the presence of degenerated germ cells in the adluminal compartment, but there were no alterations in the basal compartment. Bj-PF increased the thickness of the seminiferous epithelium and decreased the lumen diameter of the tubule. Semiquantitative histological assessment of the degree of tubule degeneration revealed that Bj-PF also increased the number of hypospermatogenic tubules compared to control. Bj-PF reduced NO2 levels in 15P-1 Sertoli cells without changing the mitochondrial metabolism. Overall, the fact that Bj-PF alters the structure and function of the seminiferous epithelium suggests that bioactive peptides found in B. jararaca snake venom can have toxicological effects on the reproductive systems of affected male mice, providing new insight into the biological characteristics of snake venom and therapeutic strategies for envenomation inflammation.

A Large-format Polyacrylamide Gel with Controllable Matrix Mechanics for Mammalian Cell Culture and Conditioned Media Production.

Tissue culture plastic has been used for routine cell culture and in vitro experiments for over 50 years. However, cells are mechanically responsive and behave differently on hard surfaces than they do on softer substrates. Polyacrylamide gels have become a popular hydrogel of choice for controlling surface stiffness and ligand density for cell adhesion. Many synthesis methods use coverslips and small gel surface areas for cell culture, which are amenable to microscopy-based experiments. However, none of the currently published methods can be scaled up to increase the surface area to accommodate conditioned media production, high volume analyte collection, or cell line expansion. To overcome this size limitation, we developed a protocol for synthesizing polyacrylamide in glass dishes using commercially available materials. This enables routine cell culture on soft surfaces and facilitates experiments that require large amounts of analyte, especially studies involving extracellular vesicles and secreted factors.

Evaluation of long-term supplementation of a direct-fed microbial and enzymatically hydrolyzed yeast cell culture product on feedlot growth performance, efficiency of dietary net energy utilization, heat stress measures, and carcass characteristics in beef steers.

The objective of this research was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and yeast cell wall (YCW) product used alone or in combination on growth performance, dietary net energy utilization, and carcass characteristics in beef steers finished under climatic conditions in the Northern Plains (NP). Single-sourced Charolais × Red Angus steers [n = 256; body weight = 246 ± 1.68 kg] were blocked by pen location in a 2 × 2 factorial arrangement of DFM and YCW. Steers were administered a series of diets common to the NP and administered ractopamine hydrochloride (RH; 300 mg/kg) during the last 28 d of the finishing phase. Steers were vaccinated and poured at processing and individually weighed on days 1, 14, 42, 77, 105, 133, 161, 182, 230, and 258. Temperature-humidity index (THI) was calculated during RH supplementation. For 98% of the experiment, the THI was lower than 72 and thus cattle were not under high-ambient temperature. On days 1, 2, 21, and 22 of RH supplementation, respiration rates (RR), and panting scores (PS) were determined before and after AM and PM feedings (0700 h, 1100 h, 1400 h, and 1700 h). A DFM + YCW interaction was noted for the proportion of steers categorized as PS 2.0 at 1100 h on day 21 (P = 0.03) and RR on day 21 at 1400 h (P = 0.02). Control steers had a greater proportion of PS 2.0 compared to DFM or YCW steers (P ≤ 0.05), while DFM + YCW steers did not differ from others (P ≥ 0.05); DFM + YCW steers had greater (P < 0.05) RR compared to DFM steers, while control and YCW steers did not differ from others (P ≥ 0.05). No DFM + YCW interactions or main effects (P ≥ 0.05) were observed for cumulative growth performance measures. However, YCW steers had 2% lower (P = 0.04) dry matter intakes compared to steers not fed YCW. No DFM + YCW interactions or main effects (P ≥ 0.05) were observed for carcass traits or liver abscess severity. However, a DFM + YCW interaction (P < 0.05) was noted for the distribution of USDA yield grade (YG) 1 and Prime carcasses. Control steers had a greater proportion (P < 0.05) of YG 1 carcasses compared to other treatments. DFM+YCW steers had a greater proportion (P < 0.05) of USDA Prime carcasses compared to DFM or YCW but were similar to control steers, which were also similar to DFM or YCW. Overall, the use of DFM and YCW alone or in combination had minimal effects on growth performance, carcass traits, and heat stress measures in steers finished in NP climatic conditions.

Biomaterial-enabled 3D cell culture technologies for extracellular vesicle manufacturing.

Extracellular vesicles (EVs) are lipid-based particles naturally released from cells and recognized as promising drug delivery vehicles for improving therapeutic outcomes. Efficient manufacturing of therapeutic EVs have been challenging for their clinical translations. Three-dimensional (3D) cell cultures enabled by biomaterial scaffolds have been used as a platform for improving EV manufacturing compared to conventional methods such as isolation from bodily fluids and standard Petri-dish cell culture. Recent studies on 3D culture derived EV production prove it to enhance the EV yield, functional cargos, and therapeutic efficacies. However, there are still challenges with scaling up 3D cell culture production platforms for industrial use. Hence, there is a high demand for designing, optimizing, and implementing large scale EV manufacturing platforms derived from 3D cell cultures. We will first review the current advances of biomaterial-enabled 3D cell cultures in EV manufacturing, followed by the effect of these 3D cell culture platforms on the EV yield, the EV quality, and therapeutic efficacies. Lastly, we will discuss the key challenges and potential for implementing biomaterial-enabled 3D culture in EV manufacturing for large scale processes in the industrial use.

Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference

Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.

Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study.

Megakaryocytes (MKs) are rare polyploid cells found in the bone marrow and produce platelets. Platelets are small cell fragments that are essential during wound healing and vascular hemostasis. In vitro differentiation of MKs from human-induced pluripotent stem cell-derived CD34+ hematopoietic stem cells (hiPSC-HSCs) could provide an alternative treatment option for thrombocytopenic patients as a platelet source. In this approach, we developed a method to produce functional MKs from hiPSC-HSCs using a xeno-free and feeder-free condition and minimize the variation and risk from animal-derived products in cell culture. We have also investigated the genome-wide expression as well as functional significance of long noncoding RNAs (lncRNAs) in hiPSC-HSC-derived MKs to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers qPCR Array Kit and identified 26 differentially regulated lncRNAs in hiPSC-HSC-derived MKs as compared with those in hiPSC-HSCs. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) was the most highly upregulated lncRNA in hiPSC-HSC-derived MKs and phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic-differentiating K562 cells. Furthermore, we have studied the potential mechanism of HOTAIRM1 based on the interactions between HOTAIRM1, p53, and miR-125b in PMA-induced K562 cells. Our results demonstrated that during MK maturation, HOTAIRM1 might be associated with the transcriptional regulation of p53 via acting as a decoy for miR-125b. Thus, the interaction between HOTAIRM1, p53, and miR-125b is likely involved in controlling cell cycling (cyclin D1), reactive oxygen species production, and apoptosis to support terminal maturation of MKs. SIGNIFICANCE STATEMENT: In vitro generation of megakaryocytes (MKs) from human-induced pluripotent stem cell-derived hematopoietic stem cells (hiPSC-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. This study has investigated the functional significance of long non-coding RNAs in hiPSC-HSC-derived MKs, which remains unclear. This study's findings suggest that the regulatory role of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in p53-mediated regulation of cyclin D1 during megakaryocytopoiesis is to promote MK maturation by decoying miR-125b.

Effects and mechanisms of animal-free hydrolysates on recombination protein yields in CHO cells.

Chinese hamster ovary (CHO) cells are the commonly used cell lines for producing recombinant therapeutic proteins (RTPs) because they possess post-translational modifications similar to human cells. Culture media are necessary for cell growth, and their quality affects the yields and quality of RTPs. Due to safety concerns for the complex purification of RTPs, the development of serum-free media (SFM) is necessary for CHO cells. To meet the need for CHO cells with higher cell density and RTP productivity with consistent product quality in large-scale suspension cultures, the optimization of SFM through adding some enzymatic animal-free hydrolysates (AFHs) is preferred. The AFHs can improve cell culture performance and product yield of RTPs without affecting their quality. Here, the effect and mechanism of various AFHs in improving CHO cell culture performance and protein expression are reviewed. KEY POINTS: • AFHs that improve the recombinant protein yield of CHO cells are reviewed. • AFHs improve recombinant protein yield via influencing cell performance. • The AFHs do not affect the quality of recombinant protein in CHO cells. • AFHs can provide nutrients, block cell cycle, and reduce oxidative stress.

Switching cell fate by the actin–auxin oscillator in Taxus: cellular aspects of plant cell fermentation

Key messagePaclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst.Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin–auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

The concept of an agroinfiltration kit for recombinant protein production for educational and commercial use-A journey through a forest of regulatory and legal implications.

Recombinant expression using Agrobacterium-mediated transient transformation (ATT) of plants has developed into a robust and versatile method to rapidly produce proteins. The capability of plants to efficiently synthesize even homo- and hetero-multimeric complex folded proteins featuring disulfide bonds and other post-translational modifications such as N-linked glycosylation makes them superior to most of the established microbial, especially prokaryotic expression hosts. Compared to production in mammalian cell cultures, ATT requires lower skills, simple technical equipment and cheaper media components. Taken together these features make the method optimally suited for R&D applications involving the development and engineering of recombinant proteins for various purposes ranging from vaccine candidates, therapeutic proteins, towards enzymes for different pharmaceutical and technical applications. Despite these advantages the technology is currently not being used outside the community of plant research. The design and realization of a kit containing all the information, instructions and ideally also the material required to perform recombinant protein production using ATT in an educational or commercial context was one of the objectives of the EU-funded Horizon 2020 project Pharma-Factory. While it is pretty straightforward to assemble a comprehensive instruction manual describing the procedure, the clarification of regulatory and legal aspects associated with the provision, dissemination and use of the different materials and organisms required to perform ATT is a complex matter. In this article, we describe the initial concept of an ATT kit for educational as well as research and development (R&D) purposes and the specific regulatory and legal implications associated with the various kit components. We cover aspects including intellectual property rights, freedom-to-operate (FTO), safety regulations for distributing genetically-modified organisms (GMOs), as well as export and import regulations. Our analysis reveals that important components of the ATT kit are freely available for research purposes but not or only with considerable effort for commercial use and distribution. We conclude with a number of considerations and requirements that need to be met in order to successfully disseminate such a kit in the future.

De-risk &amp; accelerate the drug development process for gene therapy

Kim Watanabe, PhD is the General Manager, Site Head for Patheon Translational Services. As part of the pharma services contract development and manufacturing organizational arm of Thermo Fisher Scientific, she leads the newly launched business that provides discovery stage, clinically bound clients with molecular biology, viral vector, and cell therapy services for in vivo gene therapy and ex vivo modified cell-based therapy research applications. Since joining Thermo Fisher Scientific in 2016, she has held multiple roles with increasing responsibility in product management and business integration and development. Prior to her current tenure, she was a Senior. Global Product Manager at Irvine Scientific (now Fujifilm Irvine Scientific), where she focused on cell culture products to support cell therapy applications. She was a stem cell research scientist at ScienCell Research Laboratories. Dr. Watanabe was a postdoctoral scholar at the University of California, San Diego, and received her doctoral degree in microbiology from the University of Virginia, and her BSc in cell and structural biology from the University of Illinois at Urbana-Champaign.

Engineering naturally-derived human connective tissues for clinical applications using a serum-free production system

The increasing need for tissue substitutes in reconstructive surgery spurs the development of engineering methods suited for clinical applications. Cell culture and tissue production traditionally require the use of fetal bovine serum (FBS) which is associated with various complications especially from a translational perspective. Using the self-assembly approach of tissue engineering, we hypothesized that all important parameters of tissue reconstruction can be maintained in a production system devoid of FBS from cell extraction to tissue reconstruction. We studied two commercially available serum-free medium (SFM) and xenogen-free serum-free medium (XSFM) for their impact on tissue reconstruction using human adipose-derived stem/stromal cells (ASCs) in comparison to serum-containing medium. Both media allowed higher ASC proliferation rates in primary cultures over five passages compared with 10% FBS supplemented medium while maintaining high expression of mesenchymal cell markers. For both media, we evaluated extracellular matrix production and deposition necessary to engineer manipulatable tissues using the self-assembly approach. Tissues produced in SFM exhibited a significantly increased thickness (up to 6.8-fold) compared with XSFM and FBS-containing medium. A detailed characterization of tissues produced under SFM conditions showed a substantial 50% reduction of production time without compromising key tissue features such as thickness, mechanical resistance and pro-angiogenic secretory capacities (plasminogen activator inhibitor 1, hepatocyte growth factor, vascular endothelial growth factor, angiopoietin-1) when compared to tissues produced in the control FBS-containing medium. Furthermore, we compared ASCs to the frequently used human dermal fibroblasts (DFs) in the SFM culture system. ASC-derived tissues displayed a 2.4-fold increased thickness compared to their DFs counterparts. In summary, we developed all-natural human substitutes using a production system compatible with clinical requirements. Under culture conditions devoid of bovine serum, the resulting engineered tissues displayed similar and even superior structural and functional properties over the classic FBS-containing culture conditions with a considerable 50% shortening of production time.

A polarized supercell produces specialized metabolites in cannabis trichomes

For centuries, humans have cultivated cannabis for the pharmacological properties that result from consuming its specialized metabolites, primarily cannabinoids and terpenoids. Today, cannabis is a multi-billion-dollar industry whose existence rests on the biological activity of tiny cell clusters, called glandular trichomes, found mainly on flowers. Cannabinoids are toxic to cannabis cells,1 and how the trichome cells can produce and secrete massive quantities of lipophilic metabolites is not known.1 To address this gap in knowledge, we investigated cannabis glandular trichomes using ultra-rapid cryofixation, quantitative electron microscopy, and immuno-gold labeling of cannabinoid pathway enzymes. We demonstrate that the metabolically active cells in cannabis form a "supercell," with extensive cytoplasmic bridges across the cell walls and a polar distribution of organelles adjacent to the apical surface where metabolites are secreted. The predicted metabolic role of the non-photosynthetic plastids is supported by unusual membrane arrays in the plastids and the localization of the start of the cannabinoid/terpene pathway in the stroma of the plastids. Abundant membrane contact sites connected plastid paracrystalline cores with the plastid envelope, plastid with endoplasmic reticulum (ER), and ER with plasma membrane. The final step of cannabinoid biosynthesis, catalyzed by tetrahydrocannabinolic acid synthase (THCAS), was localized in the cell-surface wall facing the extracellular storage cavity. We propose a new model of how the cannabis cells can support abundant metabolite production, with emphasis on the key role of membrane contact sites and extracellular THCA biosynthesis. This new model can inform synthetic biology approaches for cannabinoid production in yeast or cell cultures.

Interplay between crosslinking and ice nucleation controls the porous structure of freeze-dried hydrogel scaffolds.

Freeze-drying is a process of choice to texture hydrogel scaffolds with pores formed by an ice-templating mechanism. Using state-of-the-art microscopies (cryo-EBSD, μCT, CLSM), this work evidences and quantifies the effect of crosslinking and ice nucleation temperature on the porous structure of thin hydrogel scaffolds freeze-dried at a low cooling rate. We focused on a polysaccharide-based hydrogel and developed specific protocols to monitor or trigger ice nucleation for this study. At a fixed number of intermolecular crosslinks per primary molecule (p=5), the mean pore size in the dry state decreases linearly from 240 to 170μm, when ice nucleation temperature decreases from -6°C to -18°C. When ice nucleation temperature is fixed at -10°C, the mean pore size decreases from 250 to 150μm, as the crosslinking degree increases from p=3 to p=7. Scaffold infiltration ability was quantified with synthetic microspheres. The seeding efficiency was assessed with MC3T3-E1 individual cells and HepaRG™ spheroids. These data collapse into a single master curve that exhibits a sharp transition from 100% to 0%-efficiency as the entity diameter approaches the mean pore size in the dry state. Altogether, we can thus precisely tune the porosity of these 3D materials of interest for 3D cell culture and cGMP production for tissue engineering.