Project description:The development of cost-effective serum-free media is essential for the development of cell-cultured meat technologies. While some effective media have been developed for relevant cells (e.g., bovine muscle stem cells), these rely on costly recombinant albumin which is too expensive for a scalable system. In this study, we explored bulk protein isolates from rapeseed, cottonseed, and soybean as alternatives to recombinant albumin, and show that rapeseed protein isolate (RPI) is effective in replacing albumin for the serum-free culture of bovine muscle stem cells. We additionally performed proteomic characterization of the three oilseed protein isolates used in the study to compare how proteomic differences might be associated with functional differences between these proteins.
Project description:Spent culture media was collected on day 5 after in vitro fertilization from 5 human blastocysts that were subsequently transferred or frozen. Corresponding blank culture media from the same lot were cultured alongside the embryos and used as negative controls along with PBS. The samples were prepared for total RNA sequencing using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) and for small RNA sequencing using the QIAseq RNA Library Prep kit (Qiagen). Prepared libraries were sequenced as 100 bp paired end on an Illumina HiSeq 4000 sequencer for total RNA and Illumina NextSeq500 sequencer for small RNA. We found that total RNA mapped only 3% of the reads to gene regions in spent culture media and control and only 2% in PBS samples mapped to gene regions. For the small RNA analysis, human annotated miRNAs constituted from 0.1% to 1.4% of the mapped reads and 40% of small RNA reads mapped to the human genome. tiRNAs derived from 5 different mature tiRNA were differentially expressed when comparing conditioned to unconditioned media. We conclude, that in spite of applying state-of-the-art sensitive detection methods no miRNAs were found to be reliably present in the spent culture medium. In contrast, tiRNA fragments appear to be overexpressed in cultured IVF media samples.
Project description:Abstract The therapeutic properties of extracellular vesicles (EVs) derived from stem cells and stem-like cells make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies heavily on the ability to manufacture EVs in a scalable, reproducible, cGMP-compliant manner. The choice of cell culture media is a critical component for EV manufacturing. In this study, we used human amniotic epithelial cells (hAECs) as a cell model system to explore the effect of chemically defined serum-free media on EV production. Here we showed that different culture media and different cell culture supplements have variable effects on EV production including culture parameters, EV yield, and EV biogenesis. Cell viability and proliferation rate are not reliable quality indicators of EV manufacturing. The levels of common EV tetraspanins and epitope makers can be impacted by different culture media formulations even when culture parameters are maintained, and EV yield are comparable. This study has uncovered some critical aspects regarding EV production culture media that need to be considered in clinical-grade scalable EV manufacturing.
Project description:In the field of in vitro embryo production it is generally thought that the culture media are not as good as the oviductal fluid, resulting in detrimental changes of embryonic gene expression. Yet in vitro embryo culture (IVC) is one of the pillars of the assisted reproductive technologies. Therefore, unintended effects on gene expression may impact on the health of ART babies (PMID 27554442). Granted, human ART has many more components than just the culture media, but, the practice of embryo culture superimposes with the phase of life when cellular totipotency is present - that's why embryo culture media receive special attention. Discerning what culture media do is difficult because several confounders are present, including but not limited to oocyte heterogeneity, sperm heterogeneity as well as time of fertilization. We propose to take advantage of experimentally produced parthenogenetic embryos, and thereby remove two confounders (sperm variability, time of fertilization), in order to assess the effects of specific culture media of embryonic gene expression, in the mouse model of human reproduction. After synchronous parthenogenetic activation of MII oocytes, pronuclear oocytes were cultured in parallel in ART medium vs KSOM(aa) medium. Resultant blastocysts were compared and contrasted for gene expression among the parthenotes, as well as against zygotic blastocysts.