CN117187162A - HK-2 cell culture model based on microfluidic system and construction method and application thereof - Google Patents
HK-2 cell culture model based on microfluidic system and construction method and application thereof Download PDFInfo
- Publication number
- CN117187162A CN117187162A CN202311022098.6A CN202311022098A CN117187162A CN 117187162 A CN117187162 A CN 117187162A CN 202311022098 A CN202311022098 A CN 202311022098A CN 117187162 A CN117187162 A CN 117187162A
- Authority
- CN
- China
- Prior art keywords
- culture
- layer
- chip
- cells
- microfluidic system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 21
- 231100000417 nephrotoxicity Toxicity 0.000 claims abstract description 19
- 229940079593 drug Drugs 0.000 claims description 14
- 229920002799 BoPET Polymers 0.000 claims description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
- 230000010412 perfusion Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 230000002572 peristaltic effect Effects 0.000 claims description 9
- 238000011160 research Methods 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 6
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 6
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 6
- 206010029155 Nephropathy toxic Diseases 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 230000007694 nephrotoxicity Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000853 adhesive Substances 0.000 claims description 3
- 230000001070 adhesive effect Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 230000003068 static effect Effects 0.000 abstract description 36
- 230000000694 effects Effects 0.000 abstract description 19
- 238000011156 evaluation Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 87
- 239000010410 layer Substances 0.000 description 39
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 18
- 210000003734 kidney Anatomy 0.000 description 17
- 230000006870 function Effects 0.000 description 14
- 230000028327 secretion Effects 0.000 description 14
- 108091006146 Channels Proteins 0.000 description 13
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 11
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 11
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 description 11
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 description 11
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 11
- 239000012528 membrane Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 8
- 230000035699 permeability Effects 0.000 description 8
- 230000009103 reabsorption Effects 0.000 description 8
- 230000035882 stress Effects 0.000 description 8
- 210000004926 tubular epithelial cell Anatomy 0.000 description 8
- 230000004888 barrier function Effects 0.000 description 7
- 230000001186 cumulative effect Effects 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000004417 polycarbonate Substances 0.000 description 5
- 229940126680 traditional chinese medicines Drugs 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000003907 kidney function Effects 0.000 description 4
- 230000030558 renal glucose absorption Effects 0.000 description 4
- 239000003390 Chinese drug Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 206010061481 Renal injury Diseases 0.000 description 3
- 239000013553 cell monolayer Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000000512 proximal kidney tubule Anatomy 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 description 2
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 208000037806 kidney injury Diseases 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003589 nefrotoxic effect Effects 0.000 description 2
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 2
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000037978 tubular injury Diseases 0.000 description 2
- 230000010024 tubular injury Effects 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000011899 Aquaporin 2 Human genes 0.000 description 1
- 108010036221 Aquaporin 2 Proteins 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 101150071673 E6 gene Proteins 0.000 description 1
- 101150013359 E7 gene Proteins 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 108010088842 Fibrinolysin Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010072255 Integrin alpha3beta1 Proteins 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 1
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 102000005393 Sodium-Potassium-Exchanging ATPase Human genes 0.000 description 1
- 108010006431 Sodium-Potassium-Exchanging ATPase Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004082 barrier epithelial cell Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 230000004890 epithelial barrier function Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 210000001707 glomerular endothelial cell Anatomy 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000006377 glucose transport Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920005669 high impact polystyrene Polymers 0.000 description 1
- 239000004797 high-impact polystyrene Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000000885 nephron Anatomy 0.000 description 1
- 231100000381 nephrotoxic Toxicity 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 210000000557 podocyte Anatomy 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920006289 polycarbonate film Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008327 renal blood flow Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000001768 subcellular fraction Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses an HK-2 cell culture model based on a microfluidic system, and a construction method and application thereof, and belongs to the technical field of biology. The construction method comprises the steps of inoculating HK-2 cells into a microfluidic system for culture to obtain an HK-2 cell culture model based on the microfluidic system. Aiming at the problems in the evaluation of the kidney toxicity of the traditional Chinese medicine, the invention establishes an HK-2 cell culture model by utilizing a microfluidic technology and applies the model to the evaluation of the kidney toxicity of the traditional Chinese medicine. The culture model has the outstanding characteristics that not only can the cell activity and the morphology be detected, but also the renal tubular function can be detected, so that the comprehensive evaluation of the renal toxicity of the traditional Chinese medicine can be better realized. Comparing the culture model with a static transwell cell culture model of a non-microfluidic system, the culture model constructed by the invention has better sensitivity and reliability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an HK-2 cell culture model based on a microfluidic system, and a construction method and application thereof.
Background
Kidneys are the main route of excretion of drugs, and the nephrotoxic effects of drugs are closely related to the physiological properties of kidneys. These drugs are taken up, transported, accumulated, excreted in the kidneys, and in this process, the drug molecules can cause a series of reactions such as endoplasmic reticulum stress, oxidative stress, etc. to cells, causing damage to subcellular units and apoptosis and necrosis. It has been counted that 25% of patients with acute renal failure in recent years are caused by the drug renal injury. Of 162 drug varieties published in Chinese drug adverse reaction information report, the traditional Chinese drug-induced kidney injury (hereb-induced kidney injury, HIKI) accounts for 61.5% of adverse reactions caused by traditional Chinese drugs, and is mainly tubular injury. It causes kidney damage, some even acute or subacute progressive kidney function damage, which is irreversible and the renal function is not restored after withdrawal, often developing end-stage renal disease within months or 1-2 years.
The findings about kidney toxicity of traditional Chinese medicines are mostly from clinic, while the judgment of toxicity in traditional experimental research is mainly through animal experiments, and the animal experiments can comprehensively reflect the influence of medicines on organisms, but the huge reserve and compensation functions of kidneys often cover the signs of dysfunction of the kidneys, so that early diagnosis is difficult. Meanwhile, the wide development of the traditional Chinese medicine nephrotoxicity research is limited due to the long test period, large consumption, poor repeatability and the existence of species difference of animal experiments. The in vitro models currently used to evaluate renal toxicity of drugs are: in vitro renal perfusion, free nephron perfusion, kidney section, free renal cell suspension, culture of different renal cells, subcellular fraction, etc., each method has its advantages and limitations. With the development of cell biology, the kidney cell culture technology plays an important role in vitro experimental research of kidney toxicity, and particularly, the development of kidney toxicology is further promoted by gradual maturation of a culture method of kidney specific cells. Cell lines commonly used at present are pig proximal tubular epithelial cells (LLC-PK 1), dog kidney collecting tube epithelial cell line (MDCK), human proximal tubular epithelial cell line (HK-2) and the like.
HK-2 cell lines were established by Ryan et al in 1992, derived from normal adult male renal proximal tubular epithelial cells, immortalized by transfection with the E6/E7 gene of human papillomavirus (HPV-16). HK-2 cells are well differentiated and can express alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin and plasmin, and the growth is dependent on epidermal growth factor. In addition, HK-2 cells also maintain a sodium-dependent glucose transport system characteristic of tubular epithelial cells, are able to utilize and store glycogen for gluconeogenesis, and are also very sensitive to the regulation of parathyroid hormone. HK-2 cells are adult kidney cells, the functions of the HK-2 cells are more similar to those of primary cultured tubular epithelial cells, the proximal tubular epithelial cells which are the most common target cells of nephrotoxic drugs, and the HK-2 cells have become important cell models for researching the nephrotoxicity and tubular injury mechanisms of various drugs in recent years, but the application of the HK-2 cells in China is limited to the cell models established on common plastic culture plates at present.
The micro total analysis system based on the micro fluidic chip is introduced into China by Fang Zhaolun institutions for the first time in 2000, the micro fluidic chip technology is evaluated as one of 15 inventions which affect human beings in the future by the journal "Forbes", six series of articles about the micro fluidic chip are continuously published in the journal of Nature in 2006, the contents relate to all directions of single molecule measurement, cell culture, design and manufacture, industrial trend, monitoring equipment, chemical separation, synthesis and the like on the chip, and the research of the micro fluidic is pushed into a climax. Through decades of development, the current microfluidic chip not only can be used for simulating in-vivo complex microenvironments and realizing accurate regulation and control of cell or tissue culture microenvironments through effective collection of microchannels, reaction chambers and other detection functional components, but also can be used for cell or group in the chip by utilizing the mutual combination of instruments such as an optical microscope, a flow pressure sensor, an electrophoresis instrument, a chromatograph and the likeThe change of the physiological characteristics and trace basic metabolic substances is monitored in real time. The kidney organoid chip has the greatest advantage over other in vitro culture modes in that it provides a fluid flow environment for the kidney chip similar to the in vivo renal blood flow and urine flow generated after filtration. The kidney chip can detect the cell activity and also can detect the kidney function. Jang et al used a porous membrane as a scaffold for the first time in 2010 to simulate kidney collecting vessels. Placing primary rat intramedullary collecting tube cells (IMCD) on a porous membrane for applying 1dyn/cm 2 The fluid shear stress perfusion culture of (2) for 5 hours, and the functional indexes of cell activity, connection tightness, filiform actin (F-actin), E-cadherin, aquaporin 2, sodium potassium pump and the like which are related to reabsorption are measured. Musah et al designed microfluidic chips with upper and lower parallel channels and separated by a porous flexible PDMS membrane and fibronectin in the middle. Podocytes induced to differentiate by pluripotent stem cells (HIPS) are planted in the upper layer channel, primary human glomerular endothelial cells are planted in the lower layer, microfluidics is filtered from bottom to top, and the urine generation process is simulated. The chip also calculates the clearance rate by using the filtration difference value of inulin and albumin so as to verify the filtration function of the model; the damage degree of foot cells after the administration is observed when the doxorubicin is exposed to different doses, and the model can be used as a drug nephrotoxicity research platform. Homan et al 3D printed Proximal Tubule Epithelial Cells (PTEC) mimicking human kidney proximal tubule structures using 3D bioprinting techniques and perfused cultured for 65D in PDMS chips, the results showed that the extent of damage to the epithelial barrier formed by proximal tubule epithelial cells exhibited a dose-dependence when exposed to different concentrations of cyclosporin a, demonstrating the feasibility of the chip for toxicity studies. Singh et al extract from pig kidney and make natural biological ink containing intact extracellular matrix components and biochemical factors thereof, mix them with Renal Proximal Tubular Epithelial Cells (RPTECs) and HUVECS cells, squeeze print to vascular, tubular and glomerular microvascular analogs with certain hardness and integrate into microfluidic chip for culture. However, at present, no research report for evaluating the kidney toxicity of the traditional Chinese medicine by using a cell model constructed by a microfluidic system at home and abroad exists.
Disclosure of Invention
The invention aims to provide an HK-2 cell culture model based on a microfluidic system, and a construction method and application thereof, so as to solve the problems in the prior art, and establish the HK-2 cell culture system by using a microfluidic technology, wherein the culture system not only can realize the detection of cell activity and morphology, but also can realize the detection of renal tubule functions, and can be better used for omnibearing assessment of renal toxicity of traditional Chinese medicines.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a construction method of an HK-2 cell culture model based on a microfluidic system, which comprises the step of inoculating HK-2 cells into the microfluidic system for culture to obtain the HK-2 cell culture model based on the microfluidic system.
Preferably, the chip of the microfluidic system is of a five-layer four-way structure, the first layer is a chip culture cell made of PC material, the second layer is an upper layer of a PET film at the bottom of the cell culture cell, and the first layer and the second layer form an upper layer of the chip and are used for culturing HK-2 cells; the third layer is the bottom surface of the PET film of the cell culture chamber and is used for culturing HUVECs, the fourth layer is a chip runner plate made of PC material, the upper layer of the chip runner plate is a base of the chip culture chamber, the lower layer of the chip runner plate comprises a bean-shaped runner and is a flowing layer of culture solution, and the third layer and the fourth layer form a middle layer of the chip; the fifth layer is a PMMA film and forms the lower layer of the chip; and the chip flow channel plate is tightly sealed with the PMMA film, the upper layer and the middle layer of the chip are not sealed, and after each component is directly assembled, the micro peristaltic pump and the controller are connected through a perfusion pipeline system to form a five-layer four-channel structure of the chip.
Preferably, the construction method of the HK-2 cell culture model based on the microfluidic system comprises the following steps: and (3) after the bottom surface of the PET film is treated by adhesive protein, inoculating HUVECs cells, inoculating HK-2 cells on the upper surface of the PET film after 12 hours, connecting a perfusion pipeline system, adjusting the flow rate of the micro peristaltic pump, opening the controller, carrying out perfusion by taking DMEM containing 10% FBS as circulating liquid, simultaneously adding DMEM culture solution containing 10% serum into the chip culture chamber, and carrying out constant-temperature culture to obtain the HK-2 cell culture model based on a microfluidic system.
Preferably, the pod flow channels have a height of 0.25mm, 0.5mm or 1.0mm.
Preferably, the HUVECs cells are seeded at a density of 5X 10 5 The inoculation volume was 100. Mu.L/well per mL.
Preferably, the HK-2 cells are seeded at a density of 3X 10 4 Seed volume 110. Mu.L/well; the upper layer of the PET film inoculated with the HK-2 cells also contained 40. Mu.L of a DMEM medium containing 10% serum.
Preferably, the flow rate of the micro peristaltic pump is 751 mu L/min.
Preferably, the conditions of the constant temperature culture are as follows: 37 ℃,5% CO 2 Culturing for 1-7 days.
The invention also provides an HK-2 cell culture model based on the microfluidic system, which is constructed by using the construction method.
The invention also provides application of the HK-2 cell culture model based on the microfluidic system in-vitro evaluation of the renal toxicity of the drug.
The invention discloses the following technical effects:
aiming at the problems in the evaluation of the kidney toxicity of traditional Chinese medicines, a culture system of HK-2 cells is established by utilizing a microfluidic technology, the polar secretion condition of the cells, the reabsorption function of the cells on glucose and the permeability of the cells on FITC are detected within 7 days, and meanwhile, compared with a static transwell cell culture mode cultured in a non-microfluidic system, the established culture model has better sensitivity and reliability. At present, no research report on detection of kidney toxicity of traditional Chinese medicines by using the model is available at home and abroad. The culture system is applied to evaluating the renal toxicity of the medicine, and is characterized in that the renal chip not only can realize the detection of cell activity and morphology, but also can realize the detection of tubular functions, so that the renal chip can better realize the omnibearing evaluation of the renal toxicity of the traditional Chinese medicine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of HK-2 cells grown on a microporous filter membrane of an inserted petri dish;
FIG. 2 is a block diagram of a microfluidic chip for liver organs;
FIG. 3 is a line graph of the effect of microfluidic and static culture systems on GGT secretion on both sides of HK-2 cells;
FIG. 4 is a bar graph showing the effect of microfluidic and static culture systems on GGT secretion on both sides of HK-2 cells;
FIG. 5 is a line graph of the effect of microfluidic and static culture systems on ALP secretion on both sides of HK-2 cells;
FIG. 6 is a bar graph showing the effect of microfluidic and static culture systems on ALP secretion on both sides of HK-2 cells.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Human umbilical vein endothelial cells (HUVECs cells) used in the following examples were purchased from the company biotech limited, north of the city of commerce. Human proximal tubular epithelial cells (HK-2 cells) were purchased from the China academy of sciences Stem cell bank under the number SCSP-511.
Static culture adopts a standing Milicell plug-in culture dish with a pore diameter of 0.4 μm and an effective growth area of 0.6cm 2 Polycarbonate film, product of Millipore corporation.
Dynamic culture employs a microfluidic system (micromicup kit) comprising: chip flow channel PLATE (model: HZE-PLATE-01), chip culture chamber (model: HZE-WELL-01,0.4 μm aperture, effective growth area 0.17 cm) 2 ) Perfusion tubing (model: HZE-GLXT-01), micro peristaltic pump + controller (model: HZE-BMZ-01), all from Shanghai micro New technology research and development center Co.
Example 1
1. Installation of microfluidic systems
The chip has a five-layer four-channel structure. The first layer is a chip culture cell made of PC material, the second layer is the upper surface of a PET film at the bottom of the cell culture cell, and the two layers form the upper layer of the whole chip and are used for culturing HK-2 cells; the third layer is the bottom surface of the PET membrane of the cell culture chamber and is used for culturing HUVECs, the fourth layer is a chip runner plate made of PC material, the upper layer of the runner plate is a base of the chip culture chamber, the lower layer comprises bean-shaped runners, the heights of the bean-shaped runners are respectively 0.25, 0.5 and 1.0mm (marked as specifications 1, 2 and 3), and the three and four layers form the middle layer of the whole chip; the fifth layer is PMMA film and forms the lower layer of the whole chip structure. The chip flow channel plate is tightly sealed with the PMMA film, the upper layer and the middle layer are not sealed, and after each component is directly assembled, the micro peristaltic pump and the controller are connected through a perfusion pipeline system, so that a chip structure is formed. As shown in fig. 2.
2. Calculation of shear stress of microfluidic system
The shear rate value and the distribution of the shear rate value in the micro-fluidic chip structure in the flow channel are simulated by using fluid mechanics simulation software COMSOL, and the average shear rate of the whole flow channel is 122.55s -1 According to the calculation formula of Newtonian fluid shear stress, the shear stress born by the cell surface in the chip plate with the flow channel heights of 0.25, 0.5 and 1.0mm specification can be calculated to be 6.56, 1.64 and 0.41 dyne/cm 2 。
3. Establishment of culture system based on microfluidic kidney chip
3.1 microfluidic culture
After the bottom surface of the PET film was pretreated with 10. Mu.g/mL of adhesive protein in 100. Mu.L for 3 hours, the bottom surface was washed with PBS, 5X 10 5 HUVECs cells are inoculated at a density of one liter per mL, the inoculation volume is 100 mu L per hole, after 12 hours, the HUVECs cells are overturned and pressed on a chip flow channel plate which is pre-pressed with a bottom film, and the upper layer of the PET film is 3 multiplied by 10 according to the inoculation density 4 Each mL, 110. Mu.L/well of inoculation volume was inoculated with HK-2 cells, and 40. Mu.L of DMEM medium containing 10% serum was also included. Connecting a perfusion pipeline system, regulating the flow rate of a micro peristaltic pump to 751 mu L/min, opening a controller, performing perfusion by taking DMEM (DMEM with 10% FBS) as a circulating liquid, and simultaneously adding the DM containing 10% serum into chip culture cells of 3 specifications150. Mu.L of EM culture solution, 37 ℃ and 5% CO 2 Respectively culturing in an incubator for 1-7 days.
3.2 static culture
HK-2 cells were routinely cultured with DMEM medium containing 10% Fetal Bovine Serum (FBS) at 3X 10 4 The cells were inoculated into a plug-in culture dish (see FIG. 1, details of the plug-in culture dish are disclosed in the laboratory in the literature Liu Ting et al, "Early detection method of Traditional Chinese Medicine nephrotoxicity based on HK-2monolayer polar cell model cultured by transwell,2022-3-30,South Africa,2021/10244"), the cell growth support was a polycarbonate membrane (PCF membrane) having a pore size of 0.4. Mu.m, 400. Mu.L of a DMEM medium containing 10% serum and 600. Mu.L of 5% CO were added to the inside and outside of the plug-in culture dish, respectively, at 37 ℃ 2 Is cultured in an incubator for 1-7 days.
3.3 Effect of microfluidic culture and static culture System on secretion of GGT and ALP on both sides of HK-2 cells
Respectively sucking the culture solution and circulating solution (microfluidic culture) in the culture chamber after culturing for 1-7 days, and sucking the culture solution at two sides of the cells after static culture; the ALP and GGT activities were measured by using a full-automatic biochemical analyzer, and U.L was obtained -1 And (3) representing. The AP/BL ratio is calculated as follows.
AP/bl=ap side activity or content/BL side activity or content
3.4 Effect of microfluidic culture and static culture System on the permeability of HK-2 cell monolayer FITC
Cells of the above test, which had been cultured for days with cell polar secretion and reabsorption function, were subjected to FITC permeation test to confirm the monolayer barrier state of the cells. After the culture solution in each culture dish was aspirated, the culture solution was washed with a serum-free culture solution, and 1mg.mL was added to each of the AP-side microfluidic culture and the static culture system -1 600. Mu.L of the culture solution was added to the BL side of 150. Mu.L/400. Mu.L of FITC solution, and the BL side liquid was aspirated every 15min, and a new culture solution was changed. The fluorescence of the sample solution was measured at an excitation wavelength of 485nm and an emission wavelength of 510 nm. Setting different concentrations of FITC as standard curves, and calculating the accumulated leakage amount of FITC and the FITC of each square centimeter area within 2 hoursIs a cumulative percent leakage.
FITC cumulative leakage (μg/cm) 2 ) Total FITC amount and/or effective membrane area of the permeable membranes within 2h of each group
FITC cumulative percent leakage =FITC amount per total addition of total permeate membrane over 2h for each group ×100
3.5 Effect of microfluidic culture and static culture System on glucose reabsorption by HK-2 cells
HK-2 cells were pretreated and inoculated as described in 3.1-3.2, cultured with DMEM medium containing 50% FBS, and the culture medium and circulating fluid (microfluidic culture) in the culture cells were aspirated on days 1, 3, 5, and 7, respectively, and the culture fluid on both sides of the cells was aspirated as static culture; the GLU content was measured using a fully automatic biochemical analyzer for all the above samples, and the result was expressed in mmol.L -1 The AP/BL ratio is calculated as described above.
4. Test results
4.1 Effect of microfluidic culture and static culture System on secretion of GGT and ALP on both sides of HK-2 cells
As shown in tables 1, 2 and FIGS. 3-6, the test results showed that normal HK-2 cells began to form on day 3 of culture, and cell polarity was secreted mainly on the luminal side of the cells, so that the AP/BL ratio was > 1. In a microfluidic culture system, secretion of GGT and ALP both show obvious polarity; in the static culture system, only GGT presents certain polarity. Compared with a static culture system, the AP/BL ratio of the 3-runner-height microfluidic culture system GGT is obviously increased on the 5 th day of culture (P <0.05, P <0.01, P < 0.001); the AP/BL ratio of the micro-fluidic culture system ALP with the flow channel height of 0.5 and 1.0mm is obviously increased (P is less than 0.05 and P is less than 0.001) in the 4 th to 7 th days of culture, and the AP/BL ratio of the micro-fluidic culture system ALP with the flow channel height of 0.25mm is obviously increased (P is less than 0.01 and P is less than 0.001) in the 5 th to 7 th days of culture.
TABLE 1 influence of microfluidic culture and static culture System on GGT secretion on both sides of HK-2 cells (U/L, mean+ -SD, n=3)
Note that: compared with a static culture system with the same culture days, ** p＜0.01， *** p＜0.001
TABLE 2 influence of microfluidic culture and static culture System on ALP secretion on both sides of HK-2 cells (U/L, mean+ -SD, n=3)
Note that: compared with a static culture system with the same culture days, * p＜0.05， ** p＜0.01， *** p＜0.001
4.2 Effect of microfluidic culture and static culture System on FITC permeability of HK-2 cells
From the results in 4.1, it was found that HK-2 cells gradually formed on day 3 of culture, and that the polarity of the cells gradually began to develop on day 4, and that the microfluidic culture system had a significant increase in polar secretion compared to that of the static state. The experiment was performed starting from day 3 to examine the FITC permeability to verify if there was a difference in the barrier function of the epithelial cells in the two culture systems. As shown in Table 3, the results demonstrate that the FITC permeability is significantly reduced (P <0.05, P < 0.01) on days 3 to 4 of culture, regardless of the cumulative leakage amount per unit membrane area or cumulative leakage percentage, in the microfluidic group compared to the static group, indicating that cells grow well in the microfluidic system and that a dense cell monolayer structure forms earlier than in the static culture system. From day 5 to day 6 of culture, FITC permeability of both culture systems was <5%, suggesting that cells had formed a dense cell monolayer with intact barrier function, and no statistical difference was seen in the cumulative leakage of FITC for each group. By day 7 of culture, the static culture system showed a significant increase in FITC permeability, and compared with the microfluidic culture system, the cumulative leakage was significantly increased, and it was possible that some cells began to die in this culture system, but still <5%, indicating that a dense monolayer of HK-2 cells still remained, and that the growth was significantly inferior to that of HK-2 cells in the microfluidic culture system.
TABLE 3 influence of microfluidic culture and static culture System on FITC permeability of HK-2 cells (mean+ -SD, n=3)
Note that: compared with a static culture system with the same culture days, * p＜0.05， ** p＜0.01
4.3 Effect of microfluidic culture and static culture System on glucose reabsorption by HK-2 cells
As shown in table 4, the results indicate that: because the culture system is DMEM culture solution of 50% FBS, the blood sugar concentration in the whole culture system is higher and is 18.86mmoL/L, and HK-2 cells have a reabsorption function on blood sugar, so that the glucose concentration at the BL side is increased to exceed the glucose level at the AP side, and the AP/BL ratio is less than 1. In the static culture system, the ratio of glucose AP/BL is less than 1 until 3-7 days of culture, and the ratio of AP/BL is lower (P is less than 0.05 and P is less than 0.01) on days 5 and 3-5 of culture respectively when the standard of 0.5 and 1.0mm of the microfluidic culture system is compared with that of the static culture system. The HK-2 cells of the microfluidic culture system with the flow channel height of 1.0mm and 0.5mm have better glucose reabsorption function compared with the static culture system.
TABLE 4 influence of microfluidic and static culture systems on glucose reabsorption of HK-2 cells (mmoL/L, mean.+ -. SD, n=3)
Note that: compared with a static culture system with the same culture days, * p＜0.05， ** p＜0.01
4.4 Comprehensive evaluation result of 3-specification microfluidic culture system on HK-2 cell functions
From the results of 4.1 to 4.3 above, it can be seen that in 3 microfluidic culture systems, a microfluidic chip with a height of 1.0mm starts to form a good barrier function and has a reabsorption function in the third day of culture, and cell polarity is formed in the fourth day of culture; the micro-fluidic chip with the height of 0.5mm has a barrier function at the third day of culture, and the cell polarity is formed at the fourth day, but shows a reabsorption function until the fifth day of culture; the microfluidic chip with a height of 0.25mm forms a cell barrier and has a polar secretion function on the fourth day of culture, but the reabsorption function on glucose is not seen.
In summary, 3-specification microfluidic chips were evaluated for renal function, with 1.0mm better than 0.5mm better than 0.25mm, which may be compared with physiological shear stress of renal epithelial cells (0.05-1 dyne/cm 2 ) Is increasingly relevant.
Table 5 3 comprehensive evaluation results of microfluidic culture systems of various specifications on HK-2 cell function
Note that: 1) "v", "x" each indicate that the difference in function was detected to be significant or not significant in comparison with the static culture system at the corresponding culture time point. 2) "/" indicates that the system did not perform the assay at the time point of incubation.
According to the invention, by detecting the reabsorption, secretion and barrier functions of the renal tubule in a microfluidic dynamic HK-2 culture system, the culture system is proved to be superior to a static transwell cell culture mode of a non-microfluidic system, and in 3 specifications of the culture system, the whole microfluidic chip plate with the height of a runner of 1.0mm is superior to the other two specifications, so that the shear stress provided by the chip with the specifications is possibly more similar to the shear stress required by cell growth, and the microfluidic chip plate can be used as a novel detection technology for evaluating the renal toxicity of traditional Chinese medicines.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The construction method of the HK-2 cell culture model based on the microfluidic system is characterized by comprising the step of inoculating the HK-2 cells into the microfluidic system for culture to obtain the HK-2 cell culture model based on the microfluidic system.
2. The construction method of claim 1, wherein the chip of the microfluidic system has a five-layer four-way structure, the first layer is a chip culture cell made of PC material, the second layer is an upper layer of a PET film at the bottom of the cell culture cell, and the first layer and the second layer form an upper layer of the chip for culturing HK-2 cells; the third layer is the bottom surface of the PET film of the cell culture chamber and is used for culturing HUVECs, the fourth layer is a chip runner plate made of PC material, the upper layer of the chip runner plate is a base of the chip culture chamber, the lower layer of the chip runner plate comprises a bean-shaped runner and is a flowing layer of culture solution, and the third layer and the fourth layer form a middle layer of the chip; the fifth layer is a PMMA film and forms the lower layer of the chip; and the chip flow channel plate is tightly sealed with the PMMA film, the upper layer and the middle layer of the chip are not sealed, and after each component is directly assembled, the micro peristaltic pump and the controller are connected through a perfusion pipeline system to form a five-layer four-channel structure of the chip.
3. The construction method of claim 2, wherein the construction method of the microfluidic system-based HK-2 cell culture model comprises the steps of: and (3) after the bottom surface of the PET film is treated by adhesive protein, inoculating HUVECs cells, inoculating HK-2 cells on the upper surface of the PET film after 12 hours, connecting a perfusion pipeline system, adjusting the flow rate of the micro peristaltic pump, opening the controller, carrying out perfusion by taking DMEM containing 10% FBS as circulating liquid, simultaneously adding DMEM culture solution containing 10% serum into the chip culture chamber, and carrying out constant-temperature culture to obtain the HK-2 cell culture model based on a microfluidic system.
4. A method of construction according to claim 3, wherein the pod-like flow channel has a height of 0.25mm, 0.5mm or 1.0mm.
5. The method of claim 3, wherein HUVECs cells are seeded at a density of 5X 10 5 The inoculation volume was 100. Mu.L/well per mL.
6. The method of claim 3, wherein the HK-2 cells are seeded at a density of 3X 10 4 Seed volume 110. Mu.L/well; the upper layer of the PET film inoculated with the HK-2 cells also contained 40. Mu.L of a DMEM medium containing 10% serum.
7. A method of construction according to claim 3, wherein the micro peristaltic pump has a flow rate of 751 μl/min.
8. The method according to claim 3, wherein the conditions for the constant temperature culture are: 37 ℃,5% CO 2 Culturing for 1-7 days.
9. An HK-2 cell culture model based on a microfluidic system, characterized in that it is constructed by the construction method according to any one of claims 1 to 8.
10. Use of the microfluidic system-based HK-2 cell culture model according to claim 9 in research for evaluating drug nephrotoxicity in vitro.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311022098.6A CN117187162A (en) | 2023-08-15 | 2023-08-15 | HK-2 cell culture model based on microfluidic system and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311022098.6A CN117187162A (en) | 2023-08-15 | 2023-08-15 | HK-2 cell culture model based on microfluidic system and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117187162A true CN117187162A (en) | 2023-12-08 |
Family
ID=88986038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311022098.6A Pending CN117187162A (en) | 2023-08-15 | 2023-08-15 | HK-2 cell culture model based on microfluidic system and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117187162A (en) |
-
2023
- 2023-08-15 CN CN202311022098.6A patent/CN117187162A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Marrero et al. | Gut-on-a-chip: Mimicking and monitoring the human intestine | |
| Li et al. | A glass-based, continuously zonated and vascularized human liver acinus microphysiological system (vLAMPS) designed for experimental modeling of diseases and ADME/TOX | |
| Brown et al. | Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor | |
| US10481150B2 (en) | Microfluidic device for cell-based assays | |
| US20140308688A1 (en) | Human emulated response with microfluidic enhanced systems | |
| Imura et al. | A microfluidic system to evaluate intestinal absorption | |
| US8802391B2 (en) | Method for measuring the migration of cells in a channel under the influence of an analyte | |
| Volpe | Advances in cell-based permeability assays to screen drugs for intestinal absorption | |
| Rothbauer et al. | Recent advances and future applications of microfluidic live-cell microarrays | |
| Shen et al. | Organotypic and microphysiological models of liver, gut, and kidney for studies of drug metabolism, pharmacokinetics, and toxicity | |
| CN111670245B (en) | Establishing a novel multi-organ chip for differentiating iPSC-derived cells into organ equivalents | |
| Drieschner et al. | Fish-gut-on-chip: development of a microfluidic bioreactor to study the role of the fish intestine in vitro | |
| Deng et al. | A liver-on-a-chip for hepatoprotective activity assessment | |
| CN114867839A (en) | Microfluidic system for simulating lung tissue | |
| Kurth et al. | Organs-on-a-chip engineering | |
| McCloskey et al. | The modular µSiM: a mass produced, rapidly assembled, and reconfigurable platform for the study of barrier tissue models in vitro | |
| US20230272321A1 (en) | Insert chip and a system comprising same for cell culture | |
| Boul et al. | A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation | |
| US20130236972A1 (en) | Liver Sinusoid Model | |
| Busche et al. | HepaChip-MP–a twenty-four chamber microplate for a continuously perfused liver coculture model | |
| CN1948966B (en) | Micro passageway biological chip | |
| Choublier et al. | Blood–brain barrier dynamic device with uniform shear stress distribution for microscopy and permeability measurements | |
| JP2013226112A (en) | Hepatocyte culture method | |
| Doi et al. | Enhanced podocyte differentiation and changing drug toxicity sensitivity through pressure-controlled mechanical filtration stress on a glomerulus-on-a-chip | |
| Ponmozhi et al. | Models for barrier understanding in health and disease in lab-on-a-chips |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |