小鼠Müller细胞分离与培养

Xiao Liu; Luosheng Tang; Yongqing Liu

doi:10.21769/BioProtoc.2429

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Peer-reviewed

Mouse Müller Cell Isolation and Culture

小鼠Müller细胞分离与培养

XL Xiao Liu

LT Luosheng Tang

YL Yongqing Liu email

发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2429 浏览次数: 11910

评审: Karthik KrishnamurthyAnonymous reviewer(s)

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Abstract

Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells.

Keywords: Mouse (小鼠)

Müller glial cells (Müller胶质细胞)

Stem cells (干细胞)

Retinal cell isolation (视网膜细胞分离)

Primary cell culture (原代细胞培养)

Worthington Papain Kit (Worthington木瓜蛋白酶试剂盒)

Background

Müller glial cell is a major lineage in the retina that functions to maintain retinal homeostasis through synthesis of neurotrophic factors, uptake and recycle of neurotransmitters, spatial buffering of ions, and maintenance of the blood-retinal barrier (Bringmann et al., 2006; De Melo Reis et al., 2008). Müller glia serve as retinal progenitor/stem cells in fish and, to a limited extent, in birds (Vihtelic and Hyde, 2000; Fischer and Reh, 2001). But, mammalian Müller cells have lost such a capacity to regenerate the retina, though still retain certain properties of adult stem cells such as proliferation upon a retinal damage. Researches in restoring of the lost capacity of mammalian Müller cells to repair retinal damage and understanding of the underlying mechanism are undertaken in laboratories with primary cells isolated from model animal retinas. Proteolytic enzymes are widely used in Müller cell dissociation and papain is less damaging and more effective than other proteases. Sarthy and Lam developed a method for dissociation and separation of glial cells with papain digestion followed by gentle mechanical dissociation, they found that among the enzymes used for dissociating turtle retina, papain produced the least trauma (Sarthy and Lam, 1978).

Materials and Reagents

Cell culture dishes: 35 x 10 mm (Corning, catalog number: 430166 )
15 ml centrifuge tubes (Corning, catalog number: 430790 )
Tipone^® pipette tips (USA Scientific, catalog number: 1126-7810 )
5 ml pipets (Corning, Falcon^®, catalog number: 357543 )
2 ml cryopreservation vials (Corning, catalog number: 430659 )
Alcohol Prep Pads (PDI, catalog number: B33905 )
Animals: 2- to 4-week-old mice
Worthington Papain Kit (papain dissociation system) (Worthington Biochemical, catalog number: LK003150 )
Note: The components of kit include vial 1 (Sterile Earle’s Balanced Salt Solution, EBSS), vial 2 (Papain containing L-cysteine and EDTA), vial 3 (Deoxyribonuclease I, DNase), and vial 4 (Ovomucoid protease inhibitor with bovine serum albumin).
70% ethanol
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
Penicillin-streptomycin (Pen/Strep) (10,000 μg/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Cellgro^®, catalog number: 10-013-CV )
Fetal bovine serum (FBS) (GE Healthcare, HyClone^TM, catalog number: SH30071.03 )
Gelatin (Sigma-Aldrich, catalog number: G1890-100G )
0.25% trypsin ethylenediaminetetraacetic acid (EDTA) solution (Mediatech, Cellgro^®, catalog number: 25-053-CI )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
Cell culture medium (see Recipes)
0.1% gelatin solution (see Recipes)
Cell freezing medium (see Recipes)

Equipment

Pipettes (Eppendorf)
Pipet-aid (Drummond)
Single Edge Blade (Sparco, catalog number: SPR11820 )
Dissection forceps and scissors

Iris Scissors Sharp Straight (Storz Ophthalmic Instruments, catalog number: E3344 )
Castroviejo Suturing Forceps 0.12 mm (Storz Ophthalmic Instruments, catalog number: E1796 )

37 °C, 5% CO₂ cell culture incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Model 3250 )
37 °C incubator (VWR, model: 1545 )
Allegra bench-top centrifuge (Beckman Coulter, model: Allegra^® X-15R )
Lab quake rotisserie shaker (Barnstead Thermolyne LabQuake, model: 4152110 )
Inverted routine microscope (Nikon Instruments, model: Eclipse TS100 )
Stereo binocular microscope (Olympus, model: SZ40 )
Note: Mice are euthanized by carbon dioxide asphyxiation, a carbon dioxide source, regulated dispenser, and euthanasia chamber. All animal manipulations are conducted in accordance with the policies and guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) were approved by the University of Louisville, Louisville, Kentucky, USA.