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2. Hematopoietic stem cell expansion, transfection and gene editing optimization solutions
With the obvious increase in diseases such as burns and trauma, the demand for skin tissues in surgical operations has also increased accordingly. Autologous skin transplantation is often restricted, and the development of tissue-engineered skin has important clinical application value. At present, the construction of ideal artificial skin has become a major research focus in this field. Because epidermal stem cells have the ability to proliferate indefinitely, they play an important role in the research of artificial skin. There are various methods for separating and culturing epidermal stem cells. There are many markers used to identify epidermal stem cells, but some marker molecules lack specificity. It is particularly necessary to screen specific marker molecules to distinguish epidermal stem cells.
Normal skin is divided into epidermis and dermis. The epidermis is divided into 5 layers, from the inside to the outside, the basal layer, the spinous cell layer, the granular cell layer, the clear cell layer and the keratinocyte layer. Basal cells are located on the innermost basement membrane of the epidermis, while epidermal stem cells exist in the basal layer of the epidermis and the bulge of hair follicles. In most cases, epidermal stem cells are in a resting state and are stimulated by trauma or physical and chemical factors. It can be activated, proliferate and differentiate into various cell components in the epidermis, and maintain the normal epidermal structure of the skin. Because epidermal stem cells have the ability to proliferate indefinitely, they can be used in the research of tissue engineering skin. The primary culture of epidermal stem cells can use collagen to adhere quickly, so that epidermal stem cells can adhere to the wall in time. In the process of culture, to prevent contamination by pathogens, penicillin and streptomycin can be added to the tissue separation process and the complete culture medium. . At present, the identification of epidermal stem cells is mainly based on their surface-specific marker molecules. Studies have shown that CD34 can be used as a surface marker of mouse stem cells, and human hair follicles also express CD34 [1]. Keratin 14 and 15, α6 integrin [2, 3], β1 integrin [4] and CK19 [5] are all marker molecules of epidermal stem cells. The specific markers of epidermal stem cells need to be confirmed by further studies before they can be distinguished from other keratinocytes.
Epidermal stem cell culture optimization solution
(1) X-VIVO serum-free hematopoietic cell culture medium
This series is a chemically limited medium that provides a complete and balanced environment for a variety of cells including lymphokine activated killer cells (LAK), peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Such media does not contain any exogenous growth factors, substances that artificially promote cell proliferation, or undefined supplements. They do not contain any protein kinase C stimulating factors and are suitable for studying the second messenger system in human and mouse lymphocyte activation.
✍The following X-VIVO products have been registered with the FDA .
(2) UltraCulture medium
✍UltraCULTURE™ medium (12-725F) is a universal serum - free medium for culturing a variety of mammalian cell types.
✍UltraCULTURE ™ medium mainly by insulin DMEM, F-12, recombinant human, bovine transferrin and bovine serum albumin mixture (including albumin) composition .
✍The total protein concentration of UltraCULTURE™ medium is about 3mg/ml and does not contain L-glutamine. Therefore, it is necessary to add 5mL of 200mM LG (17-605) for every 500mL medium.
✍UltraCULTURE ™ medium has also been registered with the FDA .
✍Cultivate a variety of mammalian primary cells and established cell lines (including monocytes, macrophage cell lines, epithelial cells and fibroblasts;
✍UltraCULTURE medium supplemented with 5% fetal bovine serum can also be used for endothelial cell culture).
✍Promote cell fusion during hybridoma formation;
✍Promote the production of virus particles in vaccine production;
✍You can add antifreeze (12-132A) to freeze cells in a serum-free environment.
Note : UltraCULTURE™ medium may appear turbid, but it will not affect the use (contamination caused during use is not covered).
(3) Gold combination series
Gold combination- 500mL UltraCULTURE ™ (Lonza, 12-725F) +10mL
Ultroser ™ G（Pall，15950-017）+5mL L-G（17-605E）
It can be used for the cultivation and research of a variety of stem cells (mesenchymal stem cells, adipose stem cells, embryonic stem cells).
References (swipe up and down to view)
[1] Miriam Y. Kim,et al(Cell,2018).Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CART Cell Immunotherapy for Acute Myeloid Leukemia.
X-VIVO 15 adds 5% human serum, penicillin and glutamine to culture and select CD34+ T cells, transfect the T cells with the lentivirus containing CAR33 construct, knock out the CD33 gene, and target the surface antigen of AML cells , To retain the hematopoietic function of normal bone marrow progenitor cells, thereby treating acute myeloid leukemia.
[2] Justin Eyquem,et al(Nature,2017).Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumourrejection.
X-VIVO 15 adds 5% human serum and 200U/mL IL-2 to culture CD3/CD28 activated T cells, and uses CRISPR/Cas9 to target the CD19-specific CAR gene into the TARC site, which can enhance the anti-tumor effect.
[3]Jang Hwan Cho(2018).Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses.
UltraCulture adds 2mM LG, 200U/mL penicillin, 100μg/mL streptomycin, 1mM sodium pyruvate, 5mM sodium butyrate to culture the transfected HEK293 to prepare lentivirus. After collecting the virus, inoculate it to T cells cultured with X VIVO 15 Perform follow-up experiments.
[4] Nathalie Meuleman (2006).Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical a-MEM medium.
UltraCulture added 2% Ultroser G (PALL), 2mML-G, 1% antibiotic-antifungal drug to culture MSC, the number of cultured cells and the versatility of cells are better than that of α-MEM with 15% FBS.
[5] Oksana Lockridge(2018).Comparison of Hupresin® to procainamide-Sepharose for purification of butyrylcholinesterase and acetylcholinesterase
Cultivate CHO for the expression of partially glycosylated human butyrylcholinesterase;
[6]Hua-Jiang Dong(2017). The Distribution of Transplanted Umbilical Cord Mesenchymal Stem Cells in Large Blood Vessel of Rats With; used to cultivate human umbilical cord mesenchymal stem cells;
[7]Ming Zhu, (2017).Curcumin protects mesenchymal stem cells against oxidative stress-induced apoptosis via Akt/mTOR/p70S6K pathway. For the cultivation of rat-derived MSC.