The chemical name of collagenase is collagenase, which canSpecificityGeohydrolytic naturalcollagen protein3D ofHelical structureWithout damaging othersproteinAnd organization.CollagenaseChemical essenceIt is a kind of protein, therefore, it has a significant effect on temperaturePHAnd causeProtein denaturationIs very sensitive to various factors, and is vulnerable to the influence of external conditions to change its ownconformationAnd nature.
Collagenase can be divided into human body according to the way it existsendogenousCollagenase and medicinal collagenase.Human endogenous collagenase refers to the collagenase in human body, such as gingiva, contact membrane, etcEpithelial tissueAnd joint synoviumintervertebral discThis kind of collagenase exists in different degrees inside the body, and it plays an indispensable role in the decomposition of collagen in the body.Medicinal collagenase refers to the use ofPharmaceuticalsThe high-tech means of dissolving tissueClostridiumOfZymotic fluidWhite or off white obtained from extraction, purification and refining ofsterileFreeze dried powder injectionBiological agents。
Collagenase is derived from tissue dissolving spindle cellsBacillusExtracted, mainly hydrolyzedconnective tissueMedium collagen content.When the tissue to be digested is hard and contains more connective tissue or collagenTrypsinThe effect of cell dissociation is poor. At this time, collagenase can be used to dissociate cells.Collagenase is only suitable forIntercellular substanceIt has digestive effectepithelial cellsLittle impact.Therefore, it is suitable for digestion and separationFibrous propertyTissue, epithelium and cancer tissue can separate epithelial cells from collagen without damage.The commonly used dose is the final concentration 200U/ml (about 1mg/mL) or 0.03%~0.3%.Collagenase is divided into type I, II, III, IV, V andHepatocyteSpecial collagenase should be separated and digested according toOrganization typeSelect the type of collagenase.
1. Collagenase I is used for epithelium, lung, fat andadrenal glandHistiocyteSeparation of.It is used to digest the connecting part of the tissue to make it become a single cell, and is used for the separation of mammalian cells.
2. Collagenase IV contains at least 7 kindsproteaseComposition, molecular weight ranging from 68-130KD.It can digest various tissues.
3. Collagenase V contains at least 7 protease components with molecular weights ranging from 68-130KD.It can be used to separate pancreatic islet tissueTissue segregationTo form a single cell.
4. Collagenase II is applicable to liver, bone, thyroid, heart andsalivary glandsOrganization.
CalciumMagnesium ionAnd serum components will not affect the digestion of collagenase, so BSS (such as D-hanks or PBS) or serum containingCulture mediumPreparation.
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1. Cut the washed and trimmed tissue into small pieces of about 1~2mm
2. Place the tissue block inCentrifugal tube（Triangular flask）Add 30~50 times the volume of collagenase solution in, and tighten the cover (sealFlask）。
3. Put the flask into a 37 ℃ water bath or 37 ℃ incubator and shake it every 3 minutes. It is better to put the flask into a 37 ℃ constant temperature oscillating water bath.
Digestion time is related to the type of tissue. For sometumourOrganization or other comparisonDense connective tissueThe digestion time is 4~48h. For the easily digested tissues, shock digestion at 37 ℃ can be used for 15~45min, or depending on the specific situation.If the tissue mass has dispersed and lost its shape, it will become a cell mass or a single cell once shaken, which can be considered as fully digested.After the epithelial tissue is digested by collagenase, the epithelial cells haveToleranceThere may still be some cell clusters that are not completely dispersed, but clustered epithelial cells are easier to grow than dispersed single epithelial cells. Therefore, further treatment is unnecessary if there is no special need.
4. CollectionDigestive juice(Sometimes 100 meshes are required for individual tissue blocks and tissue debris not fully digestedStainless steel meshFilter), centrifugate at 1000 r/min for 5 min, remove the supernatant, and use Hanks solution orSerum free culture mediumCentrifuge rinse for 1-2 times, remove the supernatant, add culture medium to make cell suspension, and inoculateCulture flask。
