Bioreactors, scaffolds and microcarriers and in vitro meat production—current obstacles and potential solutions

In vitro meat production presents a potential viable alternative for meat consumption, which could provide the consumer with a product indistinguishable from the original, with very similar nutritional and culinary values. Indeed, the alternative products currently accessible often lack comparable nutritional value or culinary attributes to their animal-derived counterparts. This creates challenges for their global acceptance, particularly in countries where meat consumption holds cultural significance. However, while cultured meat research has been progressing rapidly in recent years, some significant obstacles still need to be overcome before its possible commercialization. Hence, this review summarizes the most current knowledge regarding the history of cultured meat, the currently used cell sources and methods used for the purpose of in vitro meat production, with particular focus on the role of bioreactors, scaffolds and microcarriers in overcoming the current obstacles. The authors put the potential microcarrier and scaffold-based solutions in a context, discussing the ways in which they can impact the way forward for the technology, including the use of considering the potential practical and societal barriers to implementing it as a viable food source worldwide

Approaches for in vitro culture of granulosa cells and ovarian follicles

The in vitro culture of ovarian follicles or cumulus-oocyte complexes (COC) is used to study the factors that regulate follicular development and may have potential use in artificial reproductive technology (ART). Before ovulation, the follicle is formed by oocyte and cell populations known as granulosa cells (GCs). These cells build the internal and external mass of the follicular wall. Oocyte growth and proliferation of the surrounding cells depend on the gap junctions between the oocyte and the GCs. Maintenance of the optimal in vitro culture system allowing for preservation of follicle architecture and granulosa-oocyte interaction may be critical for success in vitro maturation of follicles. Recently many studies have focused on a culture of GCs, which have important functions related to steroidogenesis. Granulosa cells maintained in in vitro conditions exhibit stem cell properties making it important to consider in vitro culture (IVC) methods of the GC population

Cellular Processes in Human Ovarian Follicles Are Regulated by Expression Profile of New Gene Markers—Clinical Approach

In the growing ovarian follicle, the maturing oocyte is accompanied by cumulus (CCs) and granulosa (GCs) cells. Currently, there remain many unanswered questions about the epithelial origin of these cells. Global and targeted gene transcript levels were assessed on 1, 7, 15, 30 days of culture for CCs and GCs. Detailed analysis of the genes belonging to epithelial cell-associated ontological groups allowed us to assess a total of 168 genes expressed in CCs (97 genes) and GCs (71 genes) during long-term in vitro culture. Expression changes of the analyzed genes allowed the identification of the group of genes: TGFBR3, PTGS2, PRKX, AHI1, and IL11, whose expression decreased the most and the group of ANXA3, DKK1, CCND1, STC1, CAV1, and SFRP4 genes, whose expression significantly increased. These genes’ expression indicates CCs and GCs epithelialization processes and their epithelial origin. Expression change analysis of genes involved in epithelization processes in GCs and CCs during their in vitro culture made it possible to describe the most significantly altered of the 11 genes. Detailed analysis of gene expression in these two cell populations at different time intervals confirms their ovarian surface epithelial origin. Furthermore, some gene expression profiles appear to have tumorigenic properties, suggesting that granulosa cells may play a role in cancerogenesis

Human Ovarian Granulosa Cells Isolated during an IVF Procedure Exhibit Differential Expression of Genes Regulating Cell Division and Mitotic Spindle Formation

Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups (“cell cycle arrest”, “cell cycle process”, “mitotic spindle organization”, “mitotic spindle assembly checkpoint”, “mitotic spindle assembly”, and “mitotic spindle checkpoint”) were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics

Expression Profile of New Gene Markers and Signaling Pathways Involved in Immunological Processes in Human Cumulus-Oophorus Cells

The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus–oocyte complex (COC) show evolving gene expression profiles throughout the oocytes’ lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs’ gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the “defense response to other organism”, “defense response”, “defense response to virus”, “cytokine secretion”, “cytokine production” and “cytokine-mediated signaling pathway” GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte—granulosa cells—are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h—before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: “Cell morphogenesis”, “cell differentiation” and “cell development” were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits

Expression Profile of Genes Regulating Steroid Biosynthesis and Metabolism in Human Ovarian Granulosa Cells—A Primary Culture Approach

Because of the deep involvement of granulosa cells in the processes surrounding the cycles of menstruation and reproduction, there is a great need for a deeper understanding of the ways in which they function during the various stages of those cycles. One of the main ways in which the granulosa cells influence the numerous sex associated processes is hormonal interaction. Expression of steroid sex hormones influences a range of both primary and secondary sexual characteristics, as well as regulate the processes of oogenesis, folliculogenesis, ovulation, and pregnancy. Understanding of the exact molecular mechanisms underlying those processes could not only provide us with deep insight into the regulation of the reproductive cycle, but also create new clinical advantages in detection and treatment of various diseases associated with sex hormone abnormalities. We have used the microarray approach validated by RT-qPCR, to analyze the patterns of gene expression in primary cultures of human granulosa cells at days 1, 7, 15, and 30 of said cultures. We have especially focused on genes belonging to ontology groups associated with steroid biosynthesis and metabolism, namely “Regulation of steroid biosynthesis process” and “Regulation of steroid metabolic process”. Eleven genes have been chosen, as they exhibited major change under a culture condition. Out of those, ten genes, namely STAR, SCAP, POR, SREBF1, GFI1, SEC14L2, STARD4, INSIG1, DHCR7, and IL1B, belong to both groups. Patterns of expression of those genes were analyzed, along with brief description of their functions. That analysis helped us achieve a better understanding of the exact molecular processes underlying steroid biosynthesis and metabolism in human granulosa cells