三维细胞培养和二维培养的优点.pdf

1、Reportswww.BioT325Vol.52|No.5|2012There is ample evidence that 3-D cell cultures better mimic the in vivo conditions of a multicellular organism than do 2-D cultures,in which cells adhere to glass or plastic surfaces(14).Often,the responses of cells in a 2-D culture to various cues are quite dissimi

2、lar from those of the cells in vivo or in a 3-D culture(58).This is partly because the physiology of a cell is deter-mined,in addition to the genome,by the microenvironment,including mechanical properties of the extracellular matrix(ECM)and physical and chemical anisot-ropies(2,911),which are quite

3、different in 3-D and 2-D cultures(7,12,13).Therefore,3-D cultures are preferable over 2-D for the use in a number of fields,such as studies on stem cell differentiation,tissue morpho-genesis,cancer biology,cell-virus interac-tions,and cell-based drug screening and toxicology assays(1,14,15).Still,2-

4、D cultures cultures have the advantage of presenting every cell in the same plane.This ensures that all the cells are cultured under identical conditions,including gas exchange,nutrition supply and waste removal,and permits the cells to be easily monitored,screened,and collected for further use.On t

5、he contrary,in a 3-D culture,cells located at different distances from the surface encounter different physi-ological conditions(3,8).A variety of sophisticated and expensive systems have been developed to minimize such chemical gradients(3).Another problem is the diffi-culty of using conventional m

6、icroscopes for monitoring cells immersed at different depths in a highly scattering medium(7).Both the problems were partially overcome by“on-top”cultures,in which cells are cultured in a liquid medium while being attached to a surface of a gel,while mimicking to some extent the conditions of a 3-D

7、culture(16,17).However,in such a format,cells and their progeny are not securely immobilized and may occasionally migrate into the surrounding solution.Also,before they become attached to the gel surface,cells tend to aggregate with one another and may unevenly spread over the gel surface(17).Here w

8、e report unordered 2-D arrays of eukaryotic cells that combine the advan-tages of the microenvironment of a 3-D culture with the uniformity of conditions and ease of observation characteristic of a 2-D culture.This planar 3-D culture may be of use in a number of research and applied fields requiring

9、 observation,manip-ulation,and proliferation of a large number of individual eukaryotic cells under strictly controlled conditions.Materials and methodsAdherent cellsHeLa(CCL-2;ATCC,Manassas,VA,USA),HEK-293(CRL-1573),H1299(CRL-5803),and SC-1(CRL-1404)were grown to 90%100%confluence in 25 cm2 flasks

10、and dispersed according to the ATCC protocol(www.lgcstandards-atcc.org)followed by the addition of 10 mL Dulbeccos modified Eagles medium(DMEM)supplemented with 10%FBS(PAA Laboratories,Pasching,Austria),4 mM glutamine,50 U/mL penicillin,and 50 g/mL streptomycin.Suspension cells DT40(CRL-2111)were gr

11、own in DMEM supplemented with 8%FBS,2%chicken serum(cat.no.C5405;Sigma-Aldrich,St.Louis,MO,USA),2 mM glutamine,50 M 2-mercaptoethanol,50 U/mL penicillin,and 50 g/mL streptomycin until the density of 106 cell/mL,whereas cells K-562(CCL-243)were grown in RPMI-1640 medium supplemented with 10%FBS,4 mM

12、glutamine,50 U/mL penicillin,and 50 g/mL streptomycin until the density of 0.5 106 cells/mL.For transfecting HEK-293 cells,0.3 g GFP-encoding plasmid pEGFP-C3(Clontech Laboratories,Mountain View,CA,USA)and 1 L Unifectin-56(Unifect Group,Moscow,Russia)were mixed with 225 L serum-free DMEM,incubated f

13、or 20 min at 22C,and added to a well of a 12-well plate containing 1-day-old 50%70%confluent cell layer under 0.9 mL of the complete growth medium,and the cells were grown for one more day before use.Planar arrangement of eukaryotic cells in merged hydrogels combines the advantages of 3-D and 2-D cu

14、lturesAlexander A.Gordeev,Helena V.Chetverina,and Alexander B.ChetverinInstitute of Protein Research of the Russian Academy of Sciences,Pushchino,Moscow Region,Russia BioTechniques 52:325-331(May 2012)doi 10.2144/000113861 Keywords:suspension cells;adherent cells;population studies;cell immobilizati

15、on;high-throughput screening;cell lines;cell cloningSupplementary material for this article is available at www.BioT report an unordered 2-D array of eukaryotic cells completely embedded in a 3-D matrix.Every cell is located at the same distance from the gel surface,which ensures uniformity of growt

16、h conditions and ease of observation characteristic of a 2-D culture.Each cell is firmly immobilized,and each has a unique address in the array.The cells can be rapidly screened,individually monitored during extended time periods,and cultured with the formation of spheroid microcolonies characterist

17、ic of a 3-D culture.Individual microcolonies can be extracted from the gel and further propagated,thus enabling isolation of pure cell clones from rather dense cell populations and rapid drug-free generation of stable cell lines.ReportsReportswww.BioT326Vol.52|No.5|2012Polyacrylamide(PAA)gels were c

18、ast in 14-mm-diameter,0.4-mm-deep wells,then washed and dried as reported(18).Merged gels were prepared as follows.Cells were pelleted in a centrifuge for 5 min at 200 g(or 300 g for DT40),resuspended in Dulbeccos phosphate-buffered solution(DPBS;8 mM Na2HPO4/1.5 mM KH2PO4,pH 7.5,138 mM NaCl,2.7 mM

19、KCl,0.9 mM CaCl2,0.5 mM MgCl2)to the desired concentration,and incubated for 2 min at 30C.The cell suspension was mixed(1:1,v/v)with a cooled to 30C molten 1%agarose(Type IX,Ultra-low Gelling Temperature,cat.no.A5030;Sigma-Adrich)in DPBS,and 70 L mixture were poured into a well containing dry PAA ge

20、l film attached to its bottom,with simultaneous sliding a coverslip over the well.In case of inverted merged gels,the PAA gel was attached to the coverslip.Where indicated,the slide was spun for 1 min at 100 g,25C in centrifuge 5804R(Eppendorf,Hamburg,Germany),by placing the slide with the agarose l

21、ayer facing up on the tube adapter of a bucket rotor(cat.no.A-444;Eppendorf).The agarose was then solidified at 4C for 20 min.Cells were inspected using wide-field inverted microscope DM IRE 2 with mechanical stage,integrated into the AS TP system(Leica Microsystems GmbH,Wetzlar,Germany),in either a

22、 bright field mode using the Leica modulation contrast or in a GFP fluorescent mode using the I3 filter cube(excitation filter BP 450/490,dichromatic mirror 510,suppression filter LP 515).Images were obtained using either Coolpi 4500 photo camera(Nikon Russia,Moscow,Russia)or Cascade II 512 video ca

23、mera(Photometrics,Tucson,AZ,USA).Optical sections and side views of fluorescing cells and microcolonies in the GFP fluorescent mode were obtained using confocal microscope TCS SPE DM2500 and LAS AF Version 2.1.0 build 4316 software(Leica).Where indicated,non-GFPproducing cells were stained with a br

24、oad range fluorescent ink by spotting the side of the coverslip facing the gel with a CD/DVD/BD marker(LINER 2616;Centropen,Prague,Czech Republic;www.centropen.cz/product-catalogue/for-the-office/12-3-cddvdbd-markers/38-cddvdbd-liner-2616).Microcolonies were grown in inverted merged gels whose agaro

25、se layer contained an appropriate complete growth medium with or without 0.5 mg/mL bovine skin collagen(cat.no.C4243;Sigma-Aldrich).The gels were incubated upside down in a 40-mm Petri dish at 37C and 5%CO2 under 3 mL growth medium.Microcol-onies were extracted by sucking with a disposable 70-m inne

26、r diameter glass micropipet made from a borosilicate glass capillary tubing(cat.no.BF100-50-10;Sutter Instrument Company,Novato,CA,USA)using P-97 MicropipetPuller(Sutter Instrument).The micropipet was operated with the CellTram Air micro-injector(Eppendorf Austria GmbH,Vienna,Austria)mounted on the

27、TransferMan NK 2 micromanipulator(Eppendorf)integrated into the Leica AS TP system.Results and discussionWe prepared 2-D cell arrays using merged PAA/agarose gels(1819).To this end,a suspension of cells in molten low-gelling temperature agarose was poured in a shallow well made in a microscope slide

28、 having a previously cast and dried polyacrylamide gel covalently attached.When the PAA gel swells by absorbing the liquid,it displaces cells toward the coverslip.Upon formation,the agarose gel becomes partly merged with the PAA gel and immobilizes cells concentrated on the PAA gel surface.Sometimes

29、,especially for the experiments on cell growth and cloning,we used inverted merged gels.In this case,the dry PAA matrix was covalently attached to a glass slip covering a well filled with molten agarose containing cells.To become immobilized,cells must be submerged in the agarose gel at some depth;h

30、ence,the agarose must harden before it is completely sucked up by the PAA matrix.However,in this case,cells are not layered into a monolayer;rather,they populate the entire space above the PAA surface(Figure 1A).We found that a nearly perfect monolayer can be generated by briefly spinning the slide

31、in a centrifuge while the agarose is still liquid;this forces all the cells to sediment on the swelling PAA bed.When the PAA gel was made of 7%acrylamide and 0.07%bisacryl-amide,the cells were submerged at a depth of 10 m(Figure 1B).Decreasing the acryl-amide concentration to 4%and increasing the bi

32、sacrylamide/acrylamide ratio to 1/20 increased the depth to 25 and 20 m,respectively(Supplementary Figure S1).Apparently,the lower percentage of acryl-amide makes the PAA gel more compressible during centrifugation,whereas the higher percentage of the cross-linker reduces the rate of PAA swelling.Th

33、e resulting 2-D arrangement allows all the cells within a Figure 1.HeLa cells in merged gels.Cells suspended in molten 0.5%agarose were poured into a well made in a microscopic slide,the well bottom being covered with a dry PAA gel,followed by cooling the slide on ice.Shown are confocal microscope images of fluorescently stained cells.(A)Side view of a cell layer formed without centrifugation.(B)S