Cell Culture Is No Trivial Matter (III) Digestion Method Of Adherent Cells

The Digestion Method Of Adherent Cells Describes How To Digest And Make Cells Grow Better. The Key Of Cell Culture Is Digestion. Here Are Several Methods Of Cell Digestion. One Is Enzyme Digestion. 1. Pancreatin. This Is The Most Used. The General Concentration Is 0.25-0.5%. The Time Of Digestion Varies From A Few Minutes To Dozens Of Minutes According To Cell Types, Operation Methods, Temperature And Other Factors. 0.25% Trypsin, 37 ℃ Is Generally Enough To Digest Monolayer Adherent Cells For 1-5 Minutes, And Terminate With Serum Or Complete Medium Containing Serum. 2. Collagenase. It Is Generally Used For Digestion Of Primary Cells. This Method Has Mild Effect, Less Damage To Cells, And Takes A Little Longer To Digest. The Reason Why It Is Not Recommended Is That Collagenase Is A Little Expensive And It Is Not Necessary To Cultivate Cell Lines. 2、 Ion Chelating Agent: It Does Not Destroy Cell Surface Molecules, But Only Chelates With CAMs. Therefore, If Cell Surface Molecules Are Detected, Try, Or Even Do Not Use Enzyme Digestion. 1、EDTA。 Generally, The Concentration Is About 0.02%. When Using It To Digest Cells, It Should Be Noted That It Can Significantly Affect The PH Value, And It Is Soluble Only Under Weak Alkaline Conditions, And It Cannot Be Finally Combined. Therefore, The Digested Cells Must Be Washed Once. 3、 Physical Method. Blowing Directly Or Using A Cell Scraper Can Be Used For Cells That Adhere Firmly To The Wall. Blowing And Scraping Will Cause Serious Damage To Cells. It Is Recommended Not To Use This Method Unless Absolutely Necessary. 4、 Freezing Method. The Principle Of Cell Contraction After Freezing Is Adopted To Make Cells Fall Off From The Culture Bottle. Advantages: It Has Little Damage To Cells, Does Not Need To Stop Or Wash Cells, Is Convenient, And Does Not Need To Prepare Additional Digestive Juice. It Is Especially Suitable For Those Cells That Do Not Adhere To The Wall Very Tightly And Are Particularly Delicate. Disadvantages: The Cells Often Fall Off In Small Pieces, And The Cells Will Grow A Little Clustered After Repacking. It Is Commonly Used For The Digestion Of Mesenchymal Stem Cells And DC Cells. The Specific Process Is: 1. Wash The Cells With More PBS At 4 ℃, And Then Add An Appropriate Amount Of PBS; When PBS At 4 ℃ Is Placed On The Operating Platform, The Cells Will Soon Fall Off Due To Cold Wrinkles. 3. Blow Gently, And The Cells Will Fall Off Completely. 4. Pass On The Cells In A Certain Proportion. Cell Digestion Problems: 1. Clustering And Flocculating: Adding Edta To The Digestive Juice Can Reduce The Phenomenon Of Cell Clustering, And Serum Can Stop The Effect Of Trypsin. The Better The Serum Is, The Higher The Potency Will Be. After Digestion With Trypsin, Add Serum To Stop It. If Edta Is Added To The Digestive Juice, The Digestive Juice Should Be Emptied. If The Cell Adhesion Requirements Are Not Very Strict, Centrifugation Is Generally Not Required. For Example, 4T1 Cells Are Naturally Strong In Adhering To The Wall. It Takes 10-12 Minutes To Digest With 0.5% Trypsin (including 0.1% EDTA). When Washing With PBS, Wash The Residual Medium, Add Trypsin And Digest It In The Incubator (to Avoid Cell Damage At Room Temperature And The Highest Trypsin Activity At This Temperature) Until The Cell Shrinks And Becomes Round (can Be Observed Under A Microscope) And A Few Cells Fall Off (in Quicksand Shape), And Then Immediately Discard The Trypsin (if There Are Many Fallen Cells And A Large Number Of Cell Experiments Are Required, Then Do Not Discard The Trypsin), Add The Medium And Gently Disperse It (it Cannot Be Replaced By Serum-free Medium Or PBS, Otherwise The Cells Will Gather Into Clumps Or Flocs). It Can Also Be Digested With 2% Lidocaine For 5-8 Minutes, And Then Discarded. What To Do With Indigestion? Neutralize Immediately With The Culture Medium, And Collect All Cells Into A Sterile Centrifuge Tube At 800RPM For 3 Minutes. Discard The Supernatant, Use The Full Culture To Re Suspend It, And Replace It With A New Culture Bottle To Continue The Culture. Cells In Bad Condition Will Die And Fall Off During The Culture Process, And Can Be Removed When Changing The Liquid.