Specific Operation Of 3D Cell Culture

In Vitro Three-dimensional Cell Modeling And Imaging Is A Valuable Early Step In Candidate Molecular Toxicity Assessment. 3D Cell Culture Can Largely Detect The Preclinical Drug Development Workflow, Including The Toxicity Assessment On Cells, Tissues, Organs And The Whole Organism At A Higher Biological Level. The Ability To Model And Image At These Levels Is A Powerful Tool For Molecular Assessment And Development. Individual Cells In 3D Cell Culture Provide Data About The Influence Of Molecular Subcellular On General Cell Functions, Such As Cell Growth Rate. 3D Cell Culture Is Also Used To Assess The Toxicity Of Specific Cell Types And Provide Data On Tissue And Organ Functions. 3D Cell Culture Allows Cells To Grow, And The Culture Expands In All Directions To Simulate Natural Microstructures. This Type Of Culture Provides An Impact On The Interaction Between Molecules And Cells, Which Is More Physiological Than Two-dimensional Monolayer Culture. High Resolution Imaging Of 3D Culture Provides Valuable Information On The Influence Of Molecules On Tissue Structure And Integrity. In The Early Stage Of Drug Development, Having Relevant, Reliable And Predictable Cytotoxicity Data Can Avoid The Toxicity Tests That May Lead To The Failure Of Clinical Trials, Thus Helping To Reduce The Risk Of Drug Use And Research Costs& Nbsp; Preparations Before The Experiment 1. Prepare Cell Culture Reagents 2. Put The Subpackaged Matrigel Matrix Glue From - 20 ℃ To 4 ℃ 24 Hours In Advance To Melt It Into Liquid State; Put The Sterile 1 ML Pipette Nozzle Into A Sterile 50 ML Centrifuge Tube And Place It In A - 20 ℃ Refrigerator For Precooling. Agarose Coated 96 Well Plate 3. Accurately Measure 6 ML Of Basic Culture Based On Two 10 ML Injection Glass Bottles, Add 90 Mg Of Agarose, Cover It And Put It Into A 80 ℃ Water Bath To Heat And Dissolve It For 30 Minutes; 4. After Heating, Put The Injection Bottle Into The Sterilization Pot And Sterilize At 115 ℃ For 30 Minutes; After Sterilization, Quickly Take Out The Injection Bottle And Put It Into The Ultra Clean Table. Pour The Agarose Solution In The Injection Bottle Into The Sterile Sample Adding Tank, And Use A Multi-channel Pipette To Transfer 60% μ The Amount Of L Is Added To 96 Orifice Plate. Note: Since The Agarose Solution Will Solidify At Room Temperature, It Must Be Quickly Transferred To The Ultra Clean Table And Quickly Added To The 96 Well Plate After Being Taken Out Of The Sterilization Pot. In Addition, In Order To Ensure That The Agarose Does Not Cool Down During Sample Addition, The Sample Adding Tank And 100 Should Be Sterilized At The Same Time μ L Pipette Gun Head. 5. After Adding, The 96 Well Plate Shall Be Kept Horizontal For About 30 Min To Coagulate The Agarose In The Hole. Prepare The Cell Suspension Containing Matrix Glue To Take Cells In Logarithmic Growth Phase. Take The Primary Retinal Endothelium Of Cell Systems As An Example (product No.: ACBRI 181). Count Cells After Trypsin Digestion. Adjust The Cell Suspension Concentration To 2.0 With CultureBoost Classic Cell Culture Medium × 105 Cells/mL, Standby. After The Beaker Filled With Crushed Ice Is Sprayed With Alcohol, It Is Put Into The Ultra Clean Table. The CultureBoost Classic Cell Culture Medium And The Unfrozen Matrix Glue Are Taken Out Of The Refrigerator And Placed On The Ice. Note: Since The Matrix Matrix Glue Is Solidified At Room Temperature, It Must Be Kept At A Low Temperature During The Operation. 3. Take Out The Precooled Pipette Gun Head And Place It In The Super Clean Table. According To The Calculated Amount (2.5%, V/v), Transfer 300 μ L Matrix Glue Was Added To 12 ML Complete Culture Medium And Quickly Mixed. Note: Since The Matrix Adhesive Is Solidified At Room Temperature, The Pipette Gun Head Used Also Needs To Be Precooled. Add The Cell Suspension In Step 1 (about 600 μ 50) , Make The Cell Concentration 10000 Cells/mL, Quickly Mix And Reserve; Put The Cell Suspension Into The Agarose Coated 96 Well Plate 1 Put The Cell Suspension Containing Matrigel Matrix Glue Prepared In Step 4 Above Into The Sampling Tank, And Use A Multi-channel Pipette To Suck 200 μ L Is Added To The 96 Well Plate Coated With Agarose. 2. Centrifuge With Low Temperature Centrifuge At 4 ℃ And 1000 ℃ × G. 10 Min, Seal The Periphery Of 96 Hole Plate With Sealing Film. 3. The Growth Of Human Retinal Endothelial Cells Was Relatively Slow At 3.7 And 10 Days Of Culture, And The 100 Ul Medium In The Hole Was Replaced Every 2 Days. If Drug Administration Experiment Is Required, Calculate OD Value And IC50, Prepare A 100ul Medium, And Then Suck Out The Conventional Medium And Replace It With The Drug Adding Medium After Culturing Into A Ball. Cell Systems 3D Culture References: Brown JA, Pensabene V, Markov DA, Allward V, Neely MD, Shi M, Britt CM, Hoilett OS, Yang Q, Brewer BM, Samson PC, McCawley LJ, May JM, Webb DJ, Li D, Bowman AB, Reiserer RS, Wikswo JP. Biofluids& Nbsp; 2015 Sep;& Nbsp; 9(5): 054124.  Doi: 10.1063/1.4934713Herland A, Van Der Meer AD, FitzGerald EA, Park T-E, Sleeboom JJF, Ingber DE. PLoS ONE. 2016; 11(3): E0150360. Doi: 10.1371/journal.pone.0150360.