A kind of culture system and culture method of lacrimal gland stem cells and lacrimal gland stem cells
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A culture system and cell culture technology, applied in the field of cell engineering, can solve the problems of small number and low efficiency of sorting progenitor cells
Active Publication Date: 2021-05-07
SUN YAT SEN UNIV
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[0006] Based on this, it is necessary to provide a culture system and culture method for lacrimal gland stem cells to obtain a larger number of lacrimal gland stem cells for the problems of low efficiency and small number of progenitor cell sorting progenitor cells in the existing lacrimal gland stem cell culture system and culture method, and The lacrimal gland stem cells can be stably subcultured
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Embodiment 1
[0062] A culture system for lacrimal gland stem cells, using DMEM / F12 as the base medium, adding the following components and concentrations to form the culture solution: 1% N2, 2% B27, 1% NEAA, 1% Glutamax, 10-50ng / ml EGF, 10-100ng / ml FGF10, 5-10ng / ml Wnt3A, 5-10mM Y-27632, and use Matrigel as the scaffold for three-dimensional culture. The culture medium here is called LGSCM.
Embodiment 2
[0064] A culture system for lacrimal gland stem cells, using DMEM / F12 as the base medium, adding the following components and concentrations to form the culture medium: 5-10 μM β-mercaptoethanol, 5-10 μM 2-aminoethanol, 10-20 nM sodium selenite, 50-150ng / ml heparin, 2-5ng / ml transferrin, 5-10ng / ml vitamin C, 5-10μg / ml insulin, 6.8-9.4μg / ml bovine serum albumin-oleic acid, 5-10ng / ml FGF2, 1% NEAA, 1% Glutamax, 10-50ng / ml EGF, 10-100ng / ml FGF10, 5-10ng / ml Wnt3A, 5-10mM Y-27632, and use Matrigel as the scaffold for three-dimensional culture. The culture medium here is called 9F-LGSCM
Embodiment 3
[0066] A culture method for lacrimal gland stem cells, comprising the following steps:
[0067] a) Separating and obtaining mouse lacrimal gland single cells by enzymatic digestion;
[0068] b) According to 1×10 4 The ratio of cells / well was seeded into Matrigel matrix, and cultured in the formulated lacrimal gland stem cell culture medium LGSCM. The culture environment was 37°C, 5%-10% CO 2 , after 7 days of culture, a population of sacculus stem cells formed, and 1×10 4 Ratio of passaging cells / well.
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Abstract
The invention relates to a lacrimal gland stem cell, a culture system and a culture method of the lacrimal gland stem cell, and relates to the field of cell engineering. The culture system of lacrimal gland stem cells of the present invention uses DMEM / F12 as the base medium, and adds the following components to form the culture medium: cell culture additives, non-essential amino acids, L-alanyl-L-glutamine, mitogen-activated protein Kinase / extracellular signal regulates the composition of kinase signaling pathway ligand, fibroblast growth factor, Wnt signaling pathway ligand and ROCK signaling pathway inhibitor, and uses matrigel as a three-dimensional culture support. The culture method of the present invention is as follows: the mouse lacrimal gland tissue is digested into single cells and inoculated into Matrigel, and after solidification, the lacrimal gland stem cell culture medium is added to carry out primary culture. The mouse lacrimal gland stem cells cultured by the method of the present invention have a relatively large number, are stable enough for continuous passage, and have the function of restoring lacrimal gland secretion function, and can effectively treat dry eye.
Description
technical field [0001] The invention relates to the field of cell engineering, in particular to a three-dimensional culture system and mouse lacrimal gland stem cells (LGSC) cultured by the culture system. Background technique [0002] The lacrimal gland is an important organ of the eye, mainly composed of secretory acini and secretory ducts. The lacrimal gland is regulated by sympathetic and parasympathetic nerves, most of which are cholinergic and a small part are adrenergic. The sympathetic nerves originate from the superior cervical ganglion, the parasympathetic nerves connect with the pterygopalatine and ciliary ganglia, and some sensory nerves of the lacrimal gland connect with the trigeminal nerve. There are multiple acetylcholine receptors in the lacrimal gland, including muscarinic receptors, type I and type II vasoactive intestinal peptide receptors, and norepinephrine receptors α1 and β. The lacrimal gland can secrete lysozyme, lactoferrin, lipocalin, immunoglob...
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