3D细胞培养
3D培养间充质干细胞
Dynamic perfusion bioreactor system for3D culture of rat bone marrow mesenchymal stem cells on nanohydroxyapatite/polyamide 66scaffold in vitroXu Qian,1Fang Yuan,1Zhu Zhimin,2Mo Anchun31State Key Laboratory of Oral Diseases,Sichuan University,Chengdu,610041,People’s Republic of China2Department of Prosthodontics,West China College of Stomatology,Sichuan University,Chengdu,610064,People’s Republic of China3Department of Oral implant,West China College of Stomatology,Sichuan University,Chengdu,610041,People’s Republic of ChinaReceived3September2012;revised4December2012;accepted11December2012Published online in Wiley Online Library().DOI:10.1002/jbm.b.32894Abstract:The aim of the study was to investigate the biocom-patibility and osteogenic effectiveness of the porous nanohy-droxyapatite/polyamide66(n-HA/PA66)scaffold material that was cultured with the rat bone marrow mesenchymal stem cells(rBMSCs),under the static culture condition and the dynamic perfusion culture condition in vitro,and to investi-gate whether the3D perfusion culture condition was better in provoking proliferation of rBMSCs than the3D static culture condition.The Methyl thiazolyl tetrazolium(MTT)assay,alka-line phosphatase(ALP)activity assay,Osteocalcin(OCN) assay and scanning electron microscope(SEM)were used to observe the proliferation and differentiation of rBMSCs.The samples were respectively harvested at1st,3rd,7th,14th,and 21st days and effect comparisons were made between the two of the culture conditions.The results showed that values of MTT,ALP,and OCN were increased continuously and revealed a significant difference between the two culture con-ditions(p<0.05).On the14th day,SEM revealed calcified nodules2–8l m in diameter in the lamellar structure.Under the static culture condition,the pores were covered with the cells looking like a piece of blanket,but under the perfusion culture condition the cells were observed to have a3D lamel-lar structure.In conclusion,the porous n-HA/PA66scaffold material can be used as a good candidate material for the bone scaffold construction in the tissue engineering because of its excellent3D structure,which can greatly improve the proliferation and differentiation of rBMSCs and make them proliferate and osteogenesis even better under the perfusion culture condition.V C2013Wiley Periodicals,Inc.J Biomed Mater Res Part B:Appl Biomater00B:000–000,2013.Key Words:bone graft,nanohydroxyapatite/polyamide66 (n-HA/PA66)scaffold,porosity,perfusion bioreactor,dynamic culture,tissue engineeringHow to cite this article:Qian X,Yuan F,Zhimin Z,Anchun M.2013.Dynamic perfusion bioreactor system for3D culture of rat bone marrow mesenchymal stem cells on nanohydroxyapatite/polyamide66scaffold in vitro.J Biomed Mater Res Part B2013:00B:000–000.INTRODUCTIONThe majority of factors such as dental extraction,periodontal disease,trauma and tumor can lead to some defective changes in the hard tissues.1Repair and reconstruction of the bone defects are the most difficult procedures in the dental treat-ment.Therefore,the bone grafts and the alveolar reconstruc-tion scaffold materials are widely used in the dental clinical practice.The scaffolds play an important role to direct and provide support to the growth of cells and serve as a matrix of the tissue formation during the bone tissue regeneration.This kind of role has well been recognized and the scaffolds have widely been used in the bone tissue engineering.Hydroxyapatite(HA)is an ideal bioactive material because of its excellent osteoconductivity and osteoinductivity.The composition and structure are similar to those of the natural bones,and it can be directly bonded to the bone tissues in vivo.2However,some factors such as abrasion resistance and corrosion resistance have limited its clinical application.3,4 Hence,the balance between the degradation and the regener-ation is very important,and the construction of the cells scaf-fold can provide a continuous structural support and make the bones able to bear a sufficient load in their function performances.Polyamide(PA)has good biocompatibility with the human tissues because its chemical structure is similar to that of the bone collagen protein.So,it has been widely used in the bio-materials for clinical medicine,for example,used as a surgical suture material,which has been used for nearly50years. However,the degradation of PA is difficult to control.To overcome this shortage,a novel porous nanohydroxyapatite/Correspondence to:M.Anchun;e-mail:moanchun@ or Z.Zhimin;e-mail:zzhimin@V C2013WILEY PERIODICALS,INC.1polyamide 66(n-HA/PA66)has been developed,which is syn-thesized from hydroxyapatite and polyamide,foamed by the thermal pressing and the injection molding techniques.5As our previous article stated,its great porosity and proper pore size can facilitate the cell seeding,survival,growth,prolifera-tion and differentiation in vivo.6In order to provide an enough theoretical support for its clinical application,we should per-form a further research on the n-HA/PA66scaffold in vitro .The bone tissue engineering is a promising technique,which can promote the development related to the bone scaffold materials,seed cells,culture bioreactor,and so forth.All the researches are based on a new concept of creating a similar microenvironment for the bone formation and har-vesting the biggest biological activity of the tissue engi-neered bones.7The bone marrow mesenchymal stem cells (BMSCs),also called the bone marrow stromal cells (BMSCs),have become the most potential seed cells because of their relative easiness to obtain,their amplification phenotype sta-bility,and their avoidance of excessive bone abnormal prolif-eration and avoidance of the related medical ethics dis-pute.8–10In addition,the culture bioreactors can imitate the living environment in vivo ,such as the rotating wall bioreac-tor and the perfusion bioreactor.11,12Since the biomechani-cal stimulation is an important influence factor of inducing BMSCs to differentiate into the osteoblastic cells.The bio-reactor systems can alleviate the nutrient transfer limita-tion,13upregulate the osteoblastic markers,13,14and increase the calcium deposition.15Therefore,this study was designed to investigate the reactiveness of the rBMSCs in vitro to eval-uate the biocompatibility and the osteogenic effectiveness of the n-HA/PA66scaffold material under the static culture condition and the dynamic perfusion culture condition in vitro ,and to investigate whether the dynamic culture condi-tion can better provoke the proliferation of rBMSCs in vitro .MATERIALS AND METHODSMaterials and characterizationThe porous n-HA/PA66scaffold materials (Figure 1)were synthesized from nanohydroxyapatite and polyamide,foamed by the thermal pressing and the injection moldingtechniques.5The bending strength,tensile strength and compressive strength of the composite with 64.5%n-HA content were 90–95,75–85,and 110–125MPa,respectively,which were similar to those of the natural bone (80–100,60–120,50–140MPa).5,16The elastic modulus of the com-posite was 5–7GPa,which was similar to that of the natural bone (3–25GPa).The pore size was 300-500l m in diame-ter and the porosity was 65–75%.There were 40pieces of materials to guarantee the comparability of the testing data.Mesenchymal stem cell donorAll animal procedures were performed according to the guidelines made by Animal Ethics Committee of Sichuan University.All experiments were conformed to the interna-tional guidelines on the ethical use of animals.We mini-mized the number of the rats used in the study and mini-mized their sufferings as much as possible.rBMSCs were isolated from the 4-week-old male or female Sprague Dawley (SD)rats 140–160g in weight,which had been purchased from Experimental Animal Cen-ter of Sichuan University.Isolation and culture of rBMSCsrBMSCs were harvested from the femur and the tibiae of the SD rats according to the protocol modified by Soleimani and Nadri.17The femur and the tibiae were placed in peni-cillin-streptomycin (Gibco,100U/ml)for 5minutes.Then,the bone marrow was flushed by Minimum Essential Me-dium Alpha Medium (a -MEM,Gibco,USA),which was sup-plemented by 10%(v/v)FBS (Gibco,Australia),using an 18gauge needle.Then,the cell suspension was transferred to the tissue culture flasks in a 37 C-5%CO 2incubator.The medium was poured half and the fresh medium was added 24hours later.After 48hours,the medium was replaced by the fresh medium,and the non-adherent cells were removed but the adherent cells were left for the culture in a mono-layer.The initial adherent cells were defined as Passage 0(P0)cells.The culture medium was refreshed every 3days until the adherent cells became confluent.Then,the P0cells were trypsinized (0.25%trypsin,Sigma,USA),washedandFIGURE 1.The surface topograph of the n-HA/PA66scaffold material.A:The material size was prepared as 16mm in diameter and 1.5mm in thickness for each experiment.One hole 2.5mm and three holes 1.5mm in diameter were drilled in the material,which better benefited the cul-ture medium passing through.Then,sterilization was performed by irradiation of Co 60.B:The material was magnified 10times for better obser-vation on the pores.n-HA/PA66:nanohydroxyapatite/polyamide 66.[Color figure can be viewed in the online issue,which is available at .]2QIAN ET AL.DYNAMIC PERFUSION BIOREACTOR SYSTEMplated into the other tissue culture flasks.rBMSCs were cul-tured to Passage 3(P3)for use in the following experimen-tal procedures.Flow cytometryP3rBMSCs were trypsinized and stained with monoclonal antibodies (CD44,CD29,CD45,CD11b,CD34)at room tem-perature for 30min.9,10Then the P3rBMSCs were per-formed by flow cytometry (China)analysis to identify the phenotypes before the experiments using.Mesenchymal multipotential differentiationP3rBMSCs were trypsinized,washed,and plated into the other tissue culture flasks with the osteogenic and adipo-genic inductive a -MEM medium (Gibco,USA)at a density of 2Â104cells/cm 2in a 37 C-5%CO 2incubator for the Alzarin red staining and oil red staining.Cell proliferation assayP3rBMSCs were trypsinized,washed and seeded on the 40pieces of n-HA/PA66scaffold material at a density of 5Â104cells /cm 2with osteogenic inductive medium in a 37 C-5%CO 2incubator for 24h.When the cells were adhered to the scaffold closely,they were equal separatedfor the following two culture conditions:the static culture condition (Figure 2)and the dynamic perfusion culture con-dition (Figures 3and 4),both with osteogenic inductive me-dium.Then,those sample cells underwent the following examinations:MTT assay,ALP activity,OCN detection assay,and SEM.Analysis of cell proliferation by MTT assayThe MTT assay is a colorimetric test for measuring the cell viability,cell proliferation,and cytotoxicity.The MTT substance is reduced by mitochondrial dehydrogenase in the living cells to a blue-colored formazan precipitate.This pre-cipitate can be dissolved by dimethylsulfoxide (DMSO,sigma,USA)and monitored by Thermo Scientific Varioskan Flash (Finland),a proxy for the cell proliferation.18In brief,MTT was first prepared as a stock solution of 5g/L in phosphate buffer and was filtered.MTT was added on the 1st ,3rd ,7th ,14th and 21st days respectively and dissolved by DMSO repeatedly.Then wash precipitate in the pores as much as possible and take out the scaffolds,the data were analyzed as absorbance at 570nm for the comparison between the static culture condition and the dynamic perfusion culturecondition.FIGURE 2.The stainless wire bent to fix the material to prevent its floating in the culture medium.Before rBMSCs were seeded,the material was soaked in the medium for 48h.rBMSCs:the rat bone marrow mesenchymal stem cells.[Color figure can be viewed in the online issue,which is available at.]FIGURE 3.Structure of the dynamic perfusion bioreactor.The dynamic perfusion bioreactor consisting of a peristaltic pump,a tubing circuit,a medium reservoir and a perfusion cartridge.The system was divided into two parts,that is,the medium circulation part and the gas exchange part.In the medium circulation part,the peristaltic pump perfused the medium out of the medium reservoir and,then,into the perfusion car-tridge.After that,the medium returned to the medium reservoir and experienced this circulation repeatedly.The flow velocity of the peristaltic pump was regulated at 2mL/min.In the gas exchange part,the mixed air contained 5%CO 2,21%O 2and 75%N 2according to the requirement of the cell culture.The mixed air passed through the bacterial filter at a speed of 60bubbles per minute.The safety bottle was placed at the end of the circulation part to guarantee the airway unobstructed.[Color figure can be viewed in the online issue,which is available at .]ORIGINAL RESEARCH REPORTJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B:APPLIED BIOMATERIALS |MONTH 2013VOL 00B,ISSUE 003ALP activity assayALP decomposes p-nitrobenzene phosphate (PNPP)into p-nitrophenol (PNP)under the relevant conditions,which reflects the degree of the cell activity according to the shade of yellow of PNP .The ALP activity was performed at 405nm using PNPP (Sigma,USA)as the substrate.The optical density (OD)value reflected the PNP generation at each minute.HTS 7000Plus High-Efficient Analyzer (USA)determined the OD value every 60seconds for 4times and calculated the absorbance changes (D A).According to the formula,the ALP activity (U/ml)was fig-ured out.The total protein content was determined by the Coo-massie brilliant blue method.Then,the ALP activity was expressed as U/mg (unit per mg of protein).Briefly,all the sam-ples in the static culture group and the dynamic culture group were collected respectively on the 1st ,3rd ,7th ,14th and 21st days.Cells were rinsed two times with PBS followed by trypsini-zation and then scraped into ddH 2O.All samples were followed by three cycles of freezing and thawing.The ALP activity values were determined by the method mentioned above.OCN detection assayThe OCN enzyme linked immunosorbent assay (ELISA)kits were used to detect the OCN levels.In the two culture groups,the culture medium was collected for determination of the OCN levels.The samples were harvested respectively on the 1st,3rd,7th,14th,and 21st days.Absorbance of each well was measured at 450nm by HTS 7000Plus.SEM observationThe samples were harvested respectively on the 1st,3rd,7th,14th,and 21st days under the two culture conditions.Then,the samples were soaked in 2.5%glutaraldehydeovernight,and the alcohol gradient dehydration was pre-pared for the SEM observation.The samples were sliced into two pieces to be convenient for observing the pores inside and the surface of both sides of scaffolds.Statistical analysisThere were at least 90pieces of materials to guarantee the comparability of the testing data,half for static condition and half for dynamic condition.Each 3pieces for ALP assay,MTT assay and SEM observation on 1,3,7,14,and 21days.The samples were harvested respectively.All the data were expressed as mean 6standard error (SE).Comparisons were performed by a paired two-tailed distribution test between the two culture conditions.Values of p <0.05were considered statistically significant.RESULTSCharacterization of rBMSCsCulture expanded rBMSCs grew as slender spindle-like or star-like cells [Figure 5(A)].Under permissive culture condi-tions,rBMSCs underwent osteogenic and adipogenic differ-entiation,which confirming their bona-fide MSC phenotype [Figure 5(B–D)].The majority of rBMSCs at P3were positive for CD44(99.6%)and CD29(99.8%),while a few cells were positive for CD45(4.8%),CD11b (0.6%)and CD34(0.4%)(Figure 6).It indicated that high-purity rBMSCs could be harvested and used in the next experiment.MTT assayIn Figure 7,the curves indicated a rising pattern for the MTT values of the two culture results.In spite of the MTT value on the 3th day of the static culture seemed alittleFIGURE 4.A,B:The scaffolds seeded with rBMSCs,which were separated by the bent stainless wires to prevent the cell injury due to the fric-tion between the materials.C:There were 10pieces of materials inside one perfusion culture chamber.To guarantee the comparability of the testing data,the materials located at the same position in the perfusion cartridge should be sent to the same test determination.Each 3pieces for ALP assay,MTT assay and SEM observation on 1,3,7,14and 21days.The samples were harvested respectively.ALP:alkaline phosphatase,MTT:Methyl thiazolyl tetrazolium,SEM:scanning electronic microscopy.[Color figure can be viewed in the online issue,which is available at .]4QIAN ET AL.DYNAMIC PERFUSION BIOREACTOR SYSTEMgreater than that of the dynamic perfusion culture,but no statistically significant difference was found between the two cultures (p >0.05);however,observations on the 14th and the 21st days showed that the MTT value of the dynamic culture exceeded the static culture,and there was a significant difference between the two groups (p <0.05).ALP assayFigure 8showed that on the 7th day the peak of ALP values were observed under the two culture conditions but after that time the falling trend of the values was found under the both conditions.The statistical analysis showed that there was significant difference in the ALP values according to the observations on the 3rd,7th,14th,and 21st days except for the observation on the 1st day (p <0.05).OCN assayFigure 9showed that the OCN values obtained under the two-culture conditions showed a rising trend during the ob-servation period.Under the two culture conditions,the OCN values were significantly greater on the 21st day than on the 3rd,7th,and 14th days (p <0.05).A statistically signifi-cant difference was found in the OCN values between the two-culture conditions on the 3rd day (p ¼0.0075),14th day (p ¼0.007)and 21st day (p ¼0.001);however,on the 7th day no significant difference was found between the two culture conditions (p ¼0.703).SEM observationThe SEM results from the static culture revealed that rBMSCs uniformly spread on the surface of scaffold after 24h of culture [Figure 10(A)].The pseudopods stretched on the pore walls,the pore size of which was 300–500l m in diameter,and the porosity was 65–75%.6On the 3rd day af-ter the cell seeding,the cells began to grow on the edge of the pores,proliferated well and connected with the other cells [Figure 10(B)].On the 7th day,the cells on the pore edge proliferated layer upon layer,looking like apavedFIGURE 5.Characterization of rBMSCs.A:Primary rBMSCs were slender spindle-like or star-like cells.B:Mineralized nodules formed after 14days of the mineralization inductive culture.C:rBMSCs were induced for 21days of the ontogenetic inductive culture.Mineralized nodules were reddish orange dyed by the Alzarin red staining.D:rBMSCs were induced for 5days of the adipogenic inductive culture.Lipid Droplet was red-dish dyed by oil red staining.E:rBMSCs grew well at the margin of the n-HA/PA66scaffold.The viability of rBMSCs around n-HA/PA66was strong though calcification was occasionally found.[Color figure can be viewed in the online issue,which is available at.]FIGURE 6.rBMSCs phenotype identification by flow cytometry.The majority of rBMSCs at P3were positive for CD44(99.6%)and CD29(99.8%),while a few cells were positive for CD45(4.8%),CD11b (0.6%)and CD34(0.4%).[Color figure can be viewed in the online issue,which is avail-able at .]ORIGINAL RESEARCH REPORTJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B:APPLIED BIOMATERIALS |MONTH 2013VOL 00B,ISSUE 005blanket.However,the cells at the bottom and on the walls of the pores had less nutrition supply.Therefore,the cells became smaller in number or even died [Figure 10(C)].On the 14th day,the pores were completely covered by the cells looking like a paved blanket [Figure 10(D)].The SEM results revealed that under the dynamic perfu-sion culture condition,rBMSCs were better adhered to the surface of the scaffolds.The cells spread on the rim and the inner wall of the pores after the 24-h perfusion culture.On the 3rd day,the proliferated cells spread on the deep pores and connected with each other [Figure 11(A)].rBMSCs became denser and denser in arrangement,and they were proliferated along with the porous stereoscopic structure af-ter 7days [Figure 11(B)].The cells growing vigorously for 14days remained at the bottom and on the inner wall of the pores.In the static culture group,the pores were covered by the cells looking like a paved blanket;however,in the dynamic perfusion culture group,the cells were shaped like the 3D lamellar structure which simulated to the shape of scaffold [Figure 11(C)].Majority of the pores were opened and unobstructed,so that the exchange of air and nutrients could be guaranteed.In the lamellar structure,calcified nod-ules 2-8l m in diameter could be observed [Figure 11(D)].DISCUSSIONSBMSCs are not only involved in the hematopoietic stem cell survival and differentiation but also have a multiplex differ-entiation potential.Since BMSCs are relatively few in the bone marrow,they have to be purified and cultivated to meet the bone tissue engineering requirement.The purifica-tion process usually includes the cell adherence separation,density gradient centrifugation,19immune magnetic beads separation,20and flow cytometry.21The monoclonal anti-body magnetic beads separation system and the flow cytom-etry require not only good experimental conditions but also a great amount of bone marrow;therefore,the maintaining of the BMSCs activity is relatively difficult.The gradient density centrifugation can harvest highly pure cells but the centrifugal speed and time have to be exactly adjusted in the different environmental conditions.So,in this study we used a mature method,i.e.,the cell adherence separation for amplification of BMSCs.This method had been proved effec-tive in the previous studies.BMSCs were proliferated on the walls but the hematopoietic cells were proliferated in the medium.Non-adherent cells were removed and the adher-ent cells were left for the culture in a monolayer along with the procedure of refreshing the medium.At last,BMSCs were trypsinized for 3or 4passages and they were puri-fied.22The majority of BMSCs at P3phenotype identification by flow cytometry indicated that high-purity BMSCs could be harvested and used in the experiment.The natural bone material was considered as nanocompo-site material,which was composed of collagen,apatite,and water;one-third was inorganic and two-thirds was organic.The bending strength,tensile strength and compressive strength of the porous n-HA/PA66scaffold material were 90–95,75–85,and 110–125MPa,respectively.5The porous n-HA/PA66scaffold material was close to the natural bone material that has the similar bending strength,tensile strength,and compressive strength (80–100,60–120,50–140MPa).16There-fore,this kind of composite can be well matched to that of the human bones.5PA had good biocompatibility with the human tissues,probably because of its similar chemical structure to that of the collagen protein.The polarity of the amide keys and the carboxy of PA could guide the tissue and cell growth,and promote the osteoid formation and mineralization.5PA had an excellent biological activity,biocompatibility and bone conduc-tion property.23n-HA/PA66also had excellentmechanicalFIGURE 7.The MTT values obtained under the static culture condi-tion and the dynamic perfusion culture condition for 1,3,7,14,and 21days.*p <0.05.[Color figure can be viewed in the online issue,which is available at.]FIGURE 8.The ALP values obtained under the static culture condition and the dynamic perfusion culture condition for 1,3,7,14,and 21days.*p <0.05.[Color figure can be viewed in the online issue,which is available at.]FIGURE 9.The OCN values obtained under the static culture condi-tion and the dynamic perfusion culture condition for 1,3,7,14,and 21days.*p <0.05.[Color figure can be viewed in the online issue,which is available at .]6QIAN ET AL.DYNAMIC PERFUSION BIOREACTOR SYSTEMcompatibility,and its mechanical strength came mainly from the hydrogen bond between carboxyl of n-HA and acylamino of PA66.The content of n-HA more or less affected the stability of the hydrogen bond and the crystallization rate of PA66,and thus affected the strength of n-HA/PA66.n-HA/PA66foamed by the thermal pressing and the injection molding techniques had the uniform pore size (diameter,300-500l m),which pro-vided a lasting stress-absorbent core framework.19The specific surface area of the porous n-HA/PA66scaffold had a large con-tact region,which facilitated adhesion,differentiation,prolifera-tion and metabolization of the cells.6The 3D scaffold structure effectively stimulated the osteoblast differentiationandFIGURE 10.The SEM observation on rBMSCs under the static culturecondition.FIGURE 11.The SEM observation on rBMSCs under the dynamic perfusion culture condition.ORIGINAL RESEARCH REPORTJOURNAL OF BIOMEDICAL MATERIALS RESEARCH B:APPLIED BIOMATERIALS |MONTH 2013VOL 00B,ISSUE 007benefited the osteoprogenitor cell proliferation and migra-tion.24,25Another experiment successfully proved that the3D porous scaffold effectively promoted the extracellular matrix synthesis,and its structure was similar to the natural bone structure.26After rBMSCs were seeded on the scaffolds,they were cultured under the two conditions,that is,the static culture condition and the dynamic perfusion culture condition. Under the static culture condition,rBMSCs were proliferated on the surface or out of the scaffolds because of the gravity action.27rBMSCs in the scaffolds only accounted for25%of the initially cultivated rBMSCs because the culture medium and the gas molecules could not be effectively transferred to the deep pores,so that the cell proliferation and mineral-ization only existed in the scaffold surface120–250l m in depth21;therefore,there was little or no bone formation at the centre of the scaffolds.28The structure of the3D scaf-fold met the tissue engineering requirement,but the static culture condition became an unfavorable factor.22In order to imitate the living environment in vivo,quite a few bio-reactors were developed.Glowacki et al.performed some experiments to prove the dynamic perfusion culture condi-tion able to increase the viability and the functions of BMSCs.29The cells could be seeded well-proportionally inside and outside of the scaffolds by the3D dynamic perfu-sion culture condition,which could promote oxygen and nutrients to enter the scaffolds so that the sufficient prolif-eration of the cells inside the scaffolds could be guaranteed.Many of the previous researches showed that the proper shear force and the mechanical distractive force could pro-mote the BMSCs osteogenic differentiation.There were many force application systems used for this purpose,for example,the four-point bending model,30the rotating wall bioreactor,31and the perfusion bioreactor.29The dynamic perfusion bioreactor used in this experiment perfused oxy-gen and carbon dioxide into the pores to guarantee the cul-ture medium circulation and nutrition exchanges.We chose aflow velocity of2ml/min to promote the cell proliferation and differentiation in our previous experiment because that the velocity was not so fast to cause rBMSCs away from the scaffolds.Compared with the3D static culture condition in this experiment,the3D dynamic perfusion culture condition could make the values of MTT,ALP,and OCN increase con-tinuously.However,the MTT value on the3th day of the static culture seemed a little greater than that of the dynamic perfusion culture,but observations on the14th and the21st days showed that the MTT value of the dynamic culture exceeded the static culture condition obvi-ously.The ALP activity examination could show a significant difference between the two-culture conditions on the3rd, 7th,14th,and21st days.The OCN value obtained under the dynamic perfusion culture condition was greater than that obtained under the static culture condition on the14th and 21st days.All the results indicated that the3D dynamic per-fusion culture condition could induce osteogenesis more effectively,which were similar to the results from other pre-vious researches.32The SEM results showed that under the static culture condition,the mass of the cells was shaped like a paved blanket on the scaffold surface,and most of the pores were covered by the so-called blanket.It was difficult tofind rBMSCs existing in the pores,but some dead cells were observed there.The reason for thosefindings was probably that the material exchange level was relatively low in that place,so that the cells had to proliferate on the nu-trient-rich liquid surface,forming the structure like a paved blanket.However,under the dynamic perfusion culture con-dition,rBMSCs formed a3D layer structure,and the cells could be clearly observed at the bottom or on the sides of the pore walls.Some liquid was observed to beflowing across the pores,so that the cells could obtain sufficient nutrients,which was a beneficial factor for the cell prolifer-ation.Some calcified nodules2-8l m in diameter could be found in the lamellar structure on the14th day,which revealed that porous n-HA/PA66could increase the calcium matrix deposition and the differentiation speed of the late-stage osteoblast.In addition,n-HA/PA66with65%-75% interconnected porosity had a greater calcium deposition rate than the other materials with a lower porosity.As the preosteoblast line was seeded in the scaffold that was9 mm in diameter and5mm in height,the central oxygen concentration would drop to0%on the5th day of the cul-ture[33].The cell death was observed subsequently in those areas of the scaffold.When the demineralized bone matrix scaffold was placed under the dynamic perfusion cul-ture condition,the central oxygen concentration increased to4%.Although this oxygen concentration level was still lower than that under the bulk medium condition(20%),it was still enough for prevention of the cell death.13 CONCLUSIONSThe innovative bone tissue-engineering porous n-HA/PA66 scaffold material is an ideal three-dimensional micro-struc-ture material for sufficient proliferation of rBMSCs.The po-rous n-HA/PA66scaffold material is confirmed to be a good candidate as a bone scaffold material used in the tissue en-gineering.And the dynamic perfusion culture condition can greatly improve proliferation and osteogenic effectiveness of rBMSCs on porous n-HA/PA66scaffold. ACKNOWLEDGMENTSThe authors are grateful to Ms.Li Xiaoyu at the State Key Labo-ratory of Oral Diseases(West China College of Stomatology) for her assistance in the cell culture and Dr.Zhu Zhimin(West China College of Stomatology)for her assistance in the experi-ment implementation.REFERENCES1.Mecall RA,Rosenfeld AL.The influence of residual ridge resorp-tion patterns onfixture placement and tooth position.1.Int J Periodontics Restorative Dent1991;11:8–23.2.Woodard JR,Hilldore AJ,Lan SK,Park CJ,Morgan AW,EurellJA,Clark SG,Wheeler MB,Jamison RD,Wagoner Johnson AJ.The mechanical properties and osteoconductivity of hydroxyapa-tite bone scaffolds with multi-scale porosity.Biomaterials2007;28:45–54.3.Strocchi R,Orsini G,Lezzi G,Scarano A,Rubini C,Pecora G,Piat-telli A.Bone regeneration with calcium sulfate:Evidence for8QIAN ET AL.DYNAMIC PERFUSION BIOREACTOR SYSTEM。
3D培养技术在细胞培养中的应用
3D培养技术在细胞培养中的应用作者：窦毅鹏来源：《科技资讯》2018年第03期摘要：细胞培养是研究体内细胞在体外生物学行为的重要的研究手段。
传统的细胞培养是在培养皿或培养瓶的二维平面上进行的，这与细胞在体内所处的三维生长环境有着很大的区别。
三维（3D）培养则是一种可以使细胞在体外条件下在进行三维生长的培养方法，可以更好地模拟细胞在体内的生长状况及环境。
在三维培养条件下细胞的许多生物学行为与传统的二维培养有着很大的不同，其应用领域也有更广泛的扩张，具有重要的研究意义。
本文将对三维细胞培养的发展、特点及应用进行简要的综述。
关键词：3D 细胞培养体外培养支架技术中图分类号：Q813 文献标识码：A 文章编号：1672-3791（2018）01（c）-0247-021 3D细胞培养的发展过程自从四十多年前常规真核细胞培养出现以来，支持细胞生长的最常见的物质为聚苯乙烯或玻璃，细胞在其平坦的二维表面可以进行生长。
应用这种细胞贴壁培养的方法，已经有成千上万的关于肿瘤细胞或正常细胞生物行为的研究被发表。
然而，对这些研究所基于的一个主要假设是，体外单层培养的细胞可以再现生物细胞在体内的生理学行为。
显然，在二维的玻璃或聚苯乙烯底物上生长的真核细胞并不能准确地反映出自然条件下组织中细胞的生长及与细胞外基质的准确的相互作用。
已经有研究发现，在体外培养条件下所观察到的许多复杂的生物学反应如受体表达、RNA 转录、细胞迁移和细胞凋亡等与在体内器官或组织中所观察到的并不相同。
从正常的细胞分裂、细胞增殖到细胞迁移及细胞凋亡等细胞生物学行为都是需要依赖于空间和时间的精确调控。
相比之下二维的细胞的培养方法则相对简单，忽略了这些已知的对细胞生长和组织生理学的精确调控有重要意义的参数。
这其中包括机械力的信号、细胞与细胞基质之间的信号传递以及相邻细胞微环境之间的信号沟通。
特别是在细胞间信号传递方面，许多二维培养实验未考虑不同细胞类型之间的相互作用，绝大多数培养物是单一细胞类型。
3d细胞培养原理
3d细胞培养原理
3D细胞培养原理
细胞培养是生物学研究中的重要手段之一，它可以为科学家提供大量的细胞样本，以便进行各种实验和研究。
传统的细胞培养方法是在平板上进行的，但是这种方法存在一些缺陷，比如细胞无法形成真正的三维结构，这对于某些研究来说是非常不利的。
因此，3D细胞培养方法应运而生。
3D细胞培养是一种新型的细胞培养方法，它可以让细胞在三维空间中自由生长和发育，从而形成真正的三维结构。
这种方法可以更好地模拟人体内部的环境，因此在药物研发、组织工程等领域有着广泛的应用前景。
3D细胞培养的原理是将细胞种植在一种特殊的基质中，这种基质可以模拟人体内部的环境，比如细胞外基质、胶原蛋白等。
这种基质可以提供细胞所需的营养和支持，同时也可以模拟细胞在人体内部的生长环境，从而促进细胞的生长和发育。
在3D细胞培养中，细胞可以自由生长和发育，形成各种不同的结构，比如球形、管状、片状等。
这些结构可以更好地模拟人体内部的组织结构，从而为药物研发和组织工程提供更加真实的模型。
3D细胞培养的优点不仅在于可以模拟人体内部的环境，还在于可以提供更加真实的实验结果。
传统的细胞培养方法往往只能提供一
些基本的实验结果，而3D细胞培养可以更加真实地模拟人体内部的环境，从而提供更加准确的实验结果。
3D细胞培养是一种新型的细胞培养方法，它可以让细胞在三维空间中自由生长和发育，从而形成真正的三维结构。
这种方法可以更好地模拟人体内部的环境，因此在药物研发、组织工程等领域有着广泛的应用前景。
3D细胞培养
cytoskeleton, ECM, RNA metabolism, protein metabolism, signal transduction, and other functions
Fold changes> 1.5-fold or greater. RT-PCR: GAPDH(三磷酸甘油醛脱氢酶) mRNA. The gene expression changes reflected influences of culture dimension.
定的底物，体外时，这些底物可以是其他细胞、 胶原、玻璃、或塑料等。
生长方式： 贴附生长型 悬浮生长型：血细胞、癌肿细胞 CHO （中国仓鼠卵巢）
在活体体内时各自具有其特殊的形态， 在体外培养时细胞常在形态上表现得比较
单一化，失去其在体内原有的某些特征。 体外培养贴壁细胞形态 上皮样细胞 成纤维样细胞
美国约翰·霍普金斯大学研究人员发现
通过3D细胞培养，可以弥补基于2D细 胞培养的药物研究结果和临床研究结果存 在差异的不足，有助于抗癌药物的筛选。
美国俄亥俄州立大学研究人员ห้องสมุดไป่ตู้
用3D细胞培养技术来培养包括人胚胎干 细胞和结肠癌细胞在内的许多不同类型的 人源细胞 ，来筛选抗癌药物 3D培养细胞的 药物反应更加接近体内的情况，因此，在 此基础上的药物筛选也更加可靠。
3D细胞培养
学生：董浩 导师：李红民老师
简要介绍 与2D细胞培养异同
实现方法
一、简要介绍
1.细胞培养
从动物活体体内取出组织，模拟体内生 理环境，在体外孵育培养，使之生存并增 殖。可分为：细胞培养、组织培养、器官 培养。
3D Cell Analysis说明书
欢迎关注Promega官方微信目录1. 什么是3D细胞培养 (3)2. 3D细胞培养的应用 (4)3. 3D细胞培养的分类 (5)4. 3D培养细胞的检测 (6)1）细胞健康检测 (7)• 细胞活性检测 (8)• 细胞凋亡检测 (10)• 细胞毒性检测 (12)2）代谢检测 (14)• 二核苷酸检测系统 (15)• 能量代谢检测系统 (16)• 氧化应激检测系统 (17)5. 检测仪器 (19)33D 细胞培养是能在细胞培养过程中为细胞提供一个更加接近体内生存条件的微环境的细胞培养技术。
■ 什么是3D 细胞培养?很长一段时间以来，科学家们一直依靠平板培养的2D 细胞来研究细胞和疾病的机制。
2D 细胞模型对于细胞培养和处理当然简单且经济。
然而，我们可以看到在过去的十年里，3D 细胞培养越来越受欢迎，因为它们在生理上更为相关，更能代表体内组织。
仔细思考，我们体内没有一种细胞以独立于其他细胞或组织的形式进行单层生长。
相反，大多数细胞自然存在于复杂的三维结构中，包括细胞外基质中的不同细胞类型。
众多的细胞-细胞和细胞-基质相互作用都对它们的行为有着深刻的影响。
此外，2D 单分子膜可以均匀地获得营养和氧气，而肿瘤等细胞团则不是这样。
3D 肿瘤球体更能代表体内肿瘤，与外层相比，内部细胞获得营养和氧气的机会更少，形成自然梯度。
类器官、球状体和3D 细胞模型研究在包括疾病建模和再生医学在内的许多应用中表现出了巨大的潜力。
相对于2D 模型，类器官和球状体等3D 细胞模型使我们有机会在生理学相关背景下更好地理解生物学的复杂性。
经过验证的实验方案和教育资源增强了我们对于培养和分析类器官和球状体的信心，引领3D 模型取得成功。
Quiescence2, nutrientsand assay reagentsDifferences in Cellular Responses“compound is non -toxic”“compound is toxic”4■ 为何要使用3D 培养细胞模型?监测3D 培养物的生物学变化处理iPS 细胞肿瘤活检建好的普通细胞系分化的iPS 细胞CRISPR 转染支持培养敲除一个蛋白（siRNA ）用蛋白处理表达一个蛋白用miRNA 处理小分子抑制剂物理学变化（如缺氧）可能在治疗前和/或后发生微球体培养3D Culture真皮成纤维细胞细胞工程细胞健康变化代谢变化表达变化基因组分析细胞模型越来越多地被用来了解疾病机制和药物研发治疗。
3D细胞培养和试验系统
简介哺乳动物细胞培养作为一个无价的细胞生物学工具已有几十年。
生长在扁平和僵硬的二维(2D)底物(如聚苯乙烯或玻璃)上的单层贴壁细胞已成为传统细胞培养系统的中流砥柱。
二维细胞培养的研究在扩宽我们对发育生物学、组织形态发生、疾病机制、药物发现、大批量蛋白生产、组织工程和再生医学方面的知识方面扮演了至关重要的角色。
与此同时，随着2D培养系统而来的缺陷也显现了出来，尤其是其不能模拟体内环境以提供生理相关的数据。
在体内，几乎所有组织的细胞都处在由一个复杂三维(3D)结构的细胞外基质(ECM)中，它们和相邻的细胞通过生化和机械关系产生着相互作用1。
细胞-细胞和细胞-细胞外基质间的相互作用构建成了一个用于维持组织特异性和稳态的3D通讯网络2。
细胞生命周期的关键事件通过受到周边细胞微环境的控制3。
2D培养试验中，细胞无法实现类似于体内的结构组织和连接，这使得细胞形态、存活力、增值能力、分化、基因和蛋白表达、对刺激的反应、药物代谢及一般的细胞功能受到了限制或减弱。
2D 培养的限制对临床前基于细胞的药物和毒性筛选试验的可预测性造成了影响。
超过90&的药物通过了体外临床前研究，却在随后的临床实验中未能达到预期的疗效或安全范围4。
癌症药物的失败率更高5, 12，因为2D培养系统常常无法构建有效的肿瘤生物学模型6,7。
而且，在使用2D模型进行临床前药物研发时，生物利用率和毒物学的研究严重依赖于动物模型的使用。
这一高失败率表明动物模型可能并不适用和/或并非人用治疗方案安全性评测的代表4,16,17。
为了克服这些短板，大量的3D细胞培养模型在近20年被开发了出来。
由于3D模型显著促进了癌生物学、组织工程和再生医学的研究，又得到了进一步的发展。
为此，细胞生物学家们、材料科学家们、生物医学工程师们以及其他开发更有用的模拟体内环境的3D模型来缩短2D培养与活体组织间差距的人，携手参与了多学科的研究。
越来越多的证据表明，3D培养建立起来的生理细胞-细胞与细胞-细胞外基质相互作用可以模拟天然组织的特异性，并且比传统2D培养的生理相关性更强8-10。
3D细胞培养在药物研发中的研究进展
3D细胞培养在药物研发中的研究进展新靶标的发现及发挥作用的分子与化合物的合成是药物研发的基础与重中之重，药代动力学和毒性效应是它们的作用机制。
技术的进步和学科之间的交叉渗透，使药物发现过程变得不那么繁琐反而更加简易。
生物信息学的发展使得药物在体内的代谢、作用及预后等方面可进行体外模拟，进而确定潜在的药物靶点成为可能。
应用生物信息学（结构建模）结合药物化学和细胞培养进行的体外药物检测已成为初期药物研发的主要方式，这种方法不仅有助于节省时间和成本，还有助于发现针对患者治疗的正误和有效与否。
近年，对三维细胞培养（three-dimensional cell culture，TDCC）技术最新进展的报道层出不穷，主要描述了该模型中癌细胞生长的不同物理特性和信号调控，癌细胞对药物的敏感性和如何使药物渗透至细胞，还报道了细胞对抗癌药物的敏感性受到基质性质和使用的细胞类型的影响。
业已证明，TDCC模型结合微阵列和生物信息学对于药物发现和筛选具有潜在的应用前景。
1 TDCC诱导的基因表达和药物效应候选药物在靶细胞中诱导的损伤程度是药物研发的价值体现，而安全性检测为副作用的发现提供了可能，是药物筛选的基础。
与单层细胞培养相比，TDCC 会诱导细胞基因和蛋白的差异表达，对识别新的药物靶标更具实际意义。
Li等对人神经母细胞瘤细胞SH-SY5Y进行了3D细胞培养，使用微阵列和RT-PCR分析了1766个基因的表达变化，发现不同基质特性诱导的TDCC可发生特征性变化，并强调了该研究可直接应用于药物剂量、代谢途径、药效等的检测，为个体化精准医疗提供最佳治疗结果。
另一项关于TDCC诱导的基因表达差异的综合研究是使用了对血管平滑肌细胞的9600个基因的微阵列分析。
显示在3D培养物（也称为球体）中超过77种与药物重新定位的相关基因发生过表达。
Peyton团队将TDCC技术引入平滑肌细胞的培养，结果显示TDCC中细胞外基质的力学特性可调节RhoA表达和活化，对细胞增殖具有显着影响，有助于改善抗增殖药物的使用。
(2020年整理)3D细胞培养.pptx
学海无 涯 1.倒置显微镜观察 3D 多细胞肿瘤球形态：直接将 96 孔板置于倒置显微镜下观 察即可。 2.激光共聚焦显微镜观察：用移液器小心取出孔内的肿瘤球，用 PBS 清洗 3 遍 后，采用 4%多聚甲醛固定，并用 Hoechst 33258 对细胞核进行染色，PBS 清洗 3 遍后在激光共聚焦显微镜下观察（图 3）。
2.采用低温离心机进行离心，离心条件为 4℃，1000×g，10 min。
注意：离心 96 孔板时为保持无菌，将 96 孔板的周围后，取出 96 孔板，摘下封口膜，喷洒酒精后放入培养箱内培养。整 个培养流程如图 1 所示。 4.在培养的第 3、5 和 7 天，更换孔内的 100 μL 培养基并采用倒置显微镜观察 肿瘤球的形态。
学海无 涯
3D 多细胞肿瘤球的培养
原创 2017-04-20 医生科研助手
3D 多细胞肿瘤球是在体外应用组织培养方法使肿瘤细胞以多细胞集聚 体的形式生长成为具有三维结构的球体。 与传统的 2D 贴壁细胞培养模型相比，3D 多细胞肿瘤球可以通过模拟三维细胞网 络、细胞与基质、细胞与细胞之间的相互作用，从而更加贴近肿瘤组织中相应的 病理生理特征。 因此，3D 多细胞肿瘤球培养模型已经逐渐应用于干细胞培养和分化、癌症研究、 药物和毒性筛选及组织工程等特定应用中。
虽然 3D 多细胞肿瘤球模型具有更显著的实体肿瘤生理相关性，但是与 2D 贴壁细 胞培养模型相比，获得大量相对统一的 3D 多细胞肿瘤球模型需要一系列的培养 过程和表征手段。
3D 细胞培养产品指南说明书
3D cell culture product selection guide for organoids and spheroids2Organoids and spheroids show great potential in many applications, including drug discovery, toxicology, anddisease modeling. These 3D cell models offer opportunities to better understand complex biology in a physiologically relevant context.As advances in culturing organoids and spheroids become more common, the need for cell culture guidance and product recommendations is becoming more prevalent. This selection guide is intended to give researchers a helpful starting point to facilitate the transition from 2D monolayer cultures to 3D cell models.IntroductionNeural organoid cultured on a Thermo Scientific ™ Nunclon ™Sphera ™ 6-Well Plate, which allows cells to grow with virtually no attachment. The organoid was stained with antibodies conjugated to Invitrogen ™ Alexa Fluor ™ 488 and Alexa Fluor ™ 594 dyes and imaged on an Invitrogen ™ EVOS ™ FL Auto 2 Imaging System at 10x magnification.Introduction 2 Cell sources4 Matrices and plasticware 5Media systems to support growth 6Tools for monitoring and detection 8Tools for imaging and analysis 9Key protocols and methods 10Custom biology solutions12Contents4Researchers utilize cell lines to investigate disease models of interest. Gibco ™ cell lines allow you to closely mimic the in vivo state and generate more physiologically relevant data from organoid and spheroid cultures. The table below shows a selection of our Gibco ™ primary and stem cells.C ell sourcesPhysiologically relevant tissue modelsSkin tissue model established from Gibco ™ Human Epidermal Keratinocytes (HEKa) on Thermo Scientific ™ Nunc ™ Cell Culture Insertsand Carrier Plate System.When growing 3D cell cultures, the surface you choose is essential for reproducible results.Scaffold-based systems are used to provide physical structures to support the assembly of cells into 3D models and to expand to significant numbers. Scaffold-free systems are matrix-free alternatives and are generally more adaptable to forming 3D cell models that are naturally established by endogenous adhesion molecules and extracellular matrices. Porous membrane–based systems are advantageous when polarization anddifferentiation of epithelial cells are needed in constructing 3D tissue models. Selecting the right culture platform is an important first step in developing a successful culture system for organoids and spheroids.M atrices and plasticwareScaffold-based and scaffold-free offeringsHeLa spheroid cultured in a scaffold-free Thermo Scientific ™ Nunclon ™ Sphera ™ 96U-well plate.M edia systems to support growth Growth, differentiation, and maturation of 3D cell modelsThe Gibco™ brand is the one most cited for media and reagents in peer-reviewedpublications on organoid and spheroid research.* Gibco™ products are widelyused and trusted for consistency in the growth, differentiation, and maturation of3D cell models. Using the right combination of media and growth factors is vitalto supporting the formation of disease-relevant 3D organoids and spheroidsfrom specialized cell types like stem cells or cancer cell lines.* Based on a third-party market report covering papers cited for disease modeling of organoids and spheroids with primary or stem cells as the starting cell type.6Growth factorsHigh-quality Gibco ™ growth factors are designed to give you high biological activity, high purity (95% pure), and <0.1 ng endotoxin per microgram. Our growth factors are verified with Gibco media for proven compatibility. For a list of growth factors, go to/growthfactorsA549 cells were plated at a density of5,000 cells/well on a Nunclon Sphera U-bottom plate and incubated for 24 hr in a CO 2incubator. The spheroids were then stained with Invitrogen ™ Image-iT ™ Green Hypoxia Reagent at a final concentration of 5 µM and Hoechst 33342 for 1 hr. The plate was imaged with a 10x objective using confocal mode on a Thermo Scientific ™ CellInsight ™ CX7 LZR High Content Analysis Platform. The image is from a maximum-intensity projection of 20 optical Z slices of 10 µm each.8qPCRWhen, where, and under what conditions are genes expressed? What triggers, or prevents, this expression? Scientists are discovering the surprisingly wide range of transcription and translation products, and how these different expression products determine the growth and health of an organism.TaqMan AssaysOver 1.8 million predesigned Applied Biosystems ™ TaqMan ® Gene Expression Assays covering a growing list of model species have been predesigned using long-standing bioinformatics expertise in primer and probe design. For more details, go to /taqmanT ools for monitoring and detectionGene expressionSpheroid staining using Invitrogen ™ CellROX ™ Deep Red Reagent. HeLa spheroids were pretreated with 100 μM menadione. Cells showingoxidative stress are stained red, and live-cell nuclei are stained blue.Cell healthReagents for 3D modelsEnsuring cells are maintaining the appropriate physiological morphology, markers, and activity is paramount to ensuring successful research outcomes. We have a full line of plate readers, imaging systems, and high-content analysis platforms to help you image and analyze your spheroids and organoids. These easy-to-use systems, combined with our broad portfolio of Invitrogen ™ fluorescent reagents for cellular assays, allow researchers to effectively evaluate and understand 3D cell models.Growing 3D models is a large investment in time and resources, and you need reassurance that your investment is going to give you the 3D models that you anticipate. Our visualization tools allow you to monitor the formation of your organoids and 3D models to give you confidence that you are heading in the right direction. These imaging and high-content analysis platforms have been recognized as trustworthy systems for analyzing organoid and spheroid cultures. In addition, Invitrogen ™ antibodies are validated* to help ensure specificity and reproducibility in research results.T ools for imaging and analysisVisualization of 3D cell models* The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical ordiagnostic use.HeLa cells were plated at a density of5,000 cells/well on a Nunclon Sphera U-bottom plate and incubated for 24 hr in a CO 2 incubator. The spheroids were then fixed with 4%formaldehyde and permeabilized with 0.25% Triton ™ X-100. The spheroids were blocked with 3% BSA and then stained with Ki-67 antibody conjugated to Alexa Fluor 647 dye using the Invitrogen ™ Zip Alexa Fluor ™ 647 Rapid Antibody Labeling Kit. The plate was imaged with a 10x objective using confocal mode on a CellInsight CX7 LZR High Content Analysis Platform. The image is from a maximum-intensity projection of 20 optical Z slices of 10 µm each.10There is a wide range of protocols and methods for 3D cell model formation published to date. Table 1 is a selection of the seminal publications fordifferent cell types, to help you get started on your journey to formation of 3D cell models.K ey protocols and methodsMost-cited publicationsSpheroid cell viability assay. Spheroid cell viability was evaluated using the Invitrogen ™ LIVE/DEAD ™ Cell Imaging Kit, where live cells are stained green and dead cells are stained red. Scale bar = 1,000 μm.HCT 116 (1,000 cells/well)11F ind out more at /3dmodelFor Research Use Only. Not for use in diagnostic procedures. © 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registeredtrademark of Roche Molecular Systems, Inc., used under permission and license. Triton is a trademark of Union Carbide Corporation. COL22613 0518Scientists today are continually being asked to transition their research to more physiologically relevant disease models. Whether your team needs help developing the right cell model or is interested in outsourcing steps of the research project, our CellModel Services team can help provide a custom 3D cell model tailored to your research needs. Reach out to us at /cellmodels .C ustom biology solutionsExtend your research capabilities and partner with our custom biology teamA549 cells were plated at a density of 5,000 cells/well on a Nunclon Sphera U-bottom plate and incubated for 24 hr in a CO 2 incubator. The spheroids were then stained with Hoechst 33342 for 1 hr. The plate was imaged with a 10x objective using confocal mode on a CellInsight CX7 LZR High Content Analysis Platform. The image is from a maximum-intensity projection of 20 optical Z slices of 10 µm each.A549 cells were plated at a density of 5,000 cells/well on a Nunclon Sphera U-bottom plate and incubated for 24 hr in a CO 2 incubator. The spheroids were then fixed with 4% formaldehyde and permeabilized with 0.25% Triton X-100. The spheroids were blocked with 3% BSA and then stained with Ki-67 antibody conjugated to Alexa Fluor 647 dye using the Zip Alexa Fluor 647 Rapid Antibody Labeling Kit and Hoechst 33342. The plate was imaged with a 10x objective using confocal mode on a CellInsight CX7 LZR High Content Analysis Platform. The image is from amaximum-intensity projection of 20 optical Z slices of 10 µm each.。
3D细胞培养，你了解吗？
3D细胞培养，你了解吗？欲了解更多⾎清与细胞学资讯，请关注↑↑CellMax胎⽜⾎清微信号导读通过模仿体内环境的特性，并利⽤传统的细胞培养研究⼯具，三维细胞模型提供了独特的视⾓来观察⼲细胞的⾏为、组织器官和肿瘤的发展过程。
建⽴体外三维培养模型将有助于跨越⼆维细胞培养与动物实验之间的鸿沟，有利于加速癌症⽣物学和组织⼯程领域的转化研究。
体外3D模型的关键特性就是能够模拟体内特定的细胞⾏为，使得能够精确预测组织发育和形态形成、细胞分化、药物和毒性筛选试验中基因型和/或表型对化合物的反应。
⼀些更基础的3D模型还在不使⽤基质胶底物情况下悬浮培养细胞团。
但是，多数更复杂的3D细胞培养模型都会使⽤⽔凝胶基质或固态⽀架。
⼤量的材料和制造技术被⽤于开发具有不同物理和⽣物特性的⽀架，以满⾜体内不同类型细胞的需求。
展开剩余88%3D细胞培养的主要类型⽔凝胶固体⽀架磁⼒悬浮1⽔凝胶三维培养天然的细胞外基质来源的⽔凝胶最被⼴泛⽤于体外3D细胞培养应⽤。
⽔凝胶是由交联的多聚链或复杂的天然或合成蛋⽩分⼦组成的⽹络构成。
由于含⼤量⽔，⽔凝胶具有和天然组织⾮常相似的⽣物物理学特性，因⽽可以作为⾼效的3D细胞培养基质。
⽔凝胶可以单独或和其他技术（如固体⽀架、可通透⽀持物、细胞微阵列和微流体设备）联⽤。
在3D培养系统中⽔凝胶有多种使⽤⽅法：包括为固体⽀架在内的多种细胞培养表⾯做包被，也可以将细胞包裹或夹在基质中间。
⽔凝胶基质中细胞的形态、⽣长和功能取决于⽣物物理学和⽣物化学特性，以及如通透性和基质硬度在内的物理特性。
天然来源的细胞培养⽔凝胶通常由蛋⽩和ECM成分（如胶原、层纤连蛋⽩、纤维蛋⽩、透明质酸、壳聚糖等）构成。
由于来源于天然成分，存在多种有助于多种细胞存活、增殖、功能实现和发育的内源因⼦，这些凝胶本⾝具有⽣物兼容性和⽣物活性，有利于细胞功能的完成。
细胞外基质（ECM）具有多种重要功能。
⾸先，它能够提供复杂的纳⽶级的结构蛋⽩架构（如胶原、层纤连蛋⽩和纤连蛋⽩），构建细胞微环境中的机械特性。
三维细胞培养技术培训ppt课件
3 水凝胶三维细胞培养
试剂盒组分 ① BD Matrigel ② PBS缓冲液 ③ 细胞回收液
赛哲生物®水凝胶三维细胞培养系列试剂盒（#SZCE13001~#SZCE13010） 乳腺癌系列、胃癌系列、宫颈癌系列、肝癌系列、肺癌系列 分别用于胶上培养、胶内培养
3 水凝胶三维细胞培养
胶内培养
胶上培养
Байду номын сангаас
3 水凝胶三维细胞培养
细胞处理
药物刺激： 胶上层培养液中加入刺激药物
细胞转染： 胶上培养： ① 使用sagene 3D-HG（#SC1201） 三维培养专用转染试剂 ② 使用慢病毒or 腺病毒感染 胶内培养： 使用慢病毒or 腺病毒感染
3 水凝胶三维细胞培养
细胞的回收
细胞回收液（cell recovery solution）：不含酶，能够解聚matrigel 1-2ml 细 胞 回 收 液 ， 轻 轻 刮 起 含 有 细 胞 的 凝 胶 （ 禁 止 吹 吸 ） ， 转 入
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① 什么是三维细胞培养 ② 如何实现三维细胞培养 ③ 水凝胶三维细胞培养 ④ 更多问题
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1 什么是三维细胞培养
——Kenneth M. Yamada, and Edna Cukierman. Modeling Tissue Morphogenesis and Cancer in 3D. [J]cell. 130, 601-610.
2 如何实现三维细胞培养
水凝胶三维培养：
将细胞培植在一定的细胞外基质中 , 细胞外基质（extracellularmatrix, ECM ）蛋白充当生长支架。
3d细胞培养方法
3d细胞培养方法
3D细胞培养是指在三维空间中培养细胞，其与常规的2D细胞培养不同，更能模拟体内细胞所处的环境，有利于更好地研究细胞的生理和病理特性。
其中，最简单的方法是利用培养基与聚合物之间的相互作用来形成3D结构，例如利用天然聚合物如胶原蛋白、明胶和微生物聚合物如海藻酸盐、琼脂等进行培养。
此外，还可以使用微流控芯片、生物打印、细胞自组装等技术。
总的来说，3D细胞培养技术仍处于不断发展和完善的阶段，不同的应用研究需要选择合适的3D细胞培养方法进行相应的研究。
3D细胞培养模型对肿瘤微环境研究的应用
3D细胞培养模型对肿瘤微环境研究的应用近年来，随着肿瘤治疗技术的不断进步，人们对肿瘤微环境的研究也越来越深入。
其中3D细胞培养模型是一种比较先进的研究手段，能够更真实地模拟肿瘤微环境，对于肿瘤发生、发展和治疗具有重要意义。
1. 3D细胞培养模型3D细胞培养模型是一种能够将细胞三维生长的培养模型，使得细胞可以在模拟的体外环境中生长、分化、移动和交流。
与2D细胞培养模型相比，3D细胞培养模型更接近真实生理环境，可以更好地模拟细胞在组织中的行为和反应。
在肿瘤微环境的研究中，3D细胞培养模型因其更真实的模拟肿瘤的组织结构、细胞内信号传递和细胞外基质分子相互作用等特点，被广泛应用于肿瘤的发生、发展和治疗。
2. 3D细胞培养模型在肿瘤微环境研究中的应用2.1 模拟肿瘤组织结构肿瘤是由肿瘤细胞、血管、免疫细胞和基质等多个细胞成分组成的，其中的相互作用和协同作用对于肿瘤的发生和发展至关重要。
而传统的2D细胞培养模型无法很好地模拟这些复杂的细胞相互关系，而3D细胞培养模型可以更好地模拟肿瘤的真实结构，使得研究者可以更直观地观察细胞之间的相互作用和协同作用，对于肿瘤的分子机制、信号通路和治疗研究等提供了更加可靠的基础。
2.2 模拟肿瘤基质环境肿瘤微环境中的基质是由细胞外基质分子、细胞外囊泡和细胞外基质分泌物等多个因素组成的，其影响了肿瘤的生长和转移。
3D细胞培养模型可以更好地模拟肿瘤基质环境，包括基质的结构、成分和物理性质等，从而使研究者更好地模拟肿瘤的行为和反应。
例如，研究者可以通过调节3D培养的氧份、pH值和温度等参数来模拟不同的肿瘤微环境，评估对于药物的敏感性、抗药性和转移的评估等。
2.3 模拟肿瘤的免疫调节肿瘤微环境中的免疫细胞和肿瘤细胞之间的相互作用非常复杂，包括免疫细胞的趋化、识别、杀伤和调节等多个方面，而传统的细胞培养模型通常无法很好地模拟这些复杂的交互作用。
而3D细胞培养模型可以更好地模拟免疫细胞与肿瘤细胞之间的相互作用，从而更好地评估免疫治疗的策略和疗效。
3D细胞培养
可以在更为接近体内环境的前提下研究细
胞间交互作用、细胞迁移、肿瘤和癌细胞与正
常细胞之间的交互作用，以及用于以细胞为基
础的药物检测等应用。
二、与2D细胞培养异同 1、基因表达变化 Brown University GRACE et al. 成神经肿瘤细胞 SH-SY5Y 胶原蛋白 人工基底膜 6孔板 RNA 基因芯片 14,564 genes 1,766 RT-PCR
所围绕，形成一个复杂的微小纤 维、缺口和孔洞构成的网络。其
中，氧气、激素和营养物质可以
运输传递，且废物可以过滤送走。 并且细胞能够对化学信号或其它
刺激产生反应而移动。
借助于各种填充基质（聚苯乙烯、纤维蛋
白、琼脂糖凝胶、人工基底膜、胶原蛋白等
纳米材料）在细胞培养过程中，为细胞提供
一个更加接近体内生存条件的三维环境。
美国约翰· 霍普金斯大学研究人员发现 通过3D细胞培养，可以弥补基于2D细 胞培养的药物研究结果和临床研究结果存 在差异的不足，有助于抗癌药物的筛选。 美国俄亥俄州立大学研究人员 用3D细胞培养技术来培养包括人胚胎干 细胞和结肠癌细胞在内的许多不同类型的 人源细胞 ，来筛选抗癌药物 3D培养细胞的 药物反应更加接近体内的情况，因此，在 此基础上的药物筛选也更加可靠。
Fold changes> 1.5-fold or greater. RT-PCR: GAPDH(三磷酸甘油醛脱氢酶) mRNA. The gene expression changes reflected influences of culture dimension.
2. 细胞形态学变化 Brown University GRACE et al. 成神经肿瘤细胞 SH-SY5Y 胶原蛋白 人工基底膜 6孔板 细胞体、轴突形态改变
3D细胞培养.pptx
本文利用 Liquid Overlay 的制备方法，以乳腺癌细胞 MDA-MB-231 和 MCF-7 为模 型制备 3D 多细胞肿瘤球并采用倒置显微镜、激光共聚焦显微镜和环境扫描电镜 对其进行详细表征。
5.若要采用 3D 多细胞肿瘤球进行药物试验，在培养 7 天后，用移液器取出孔内 的 100 μL 培养基，加入 100 μL 给药溶液，然后置于培养箱内培养并定期采用 倒置显微镜观察肿瘤球的生长状况（图 2）。 5 3D 多细胞肿瘤球的表征
4
书山有 路 1.倒置显微镜观察 3D 多细胞肿瘤球形态：直接将 96 孔板置于倒置显微镜下观 察即可。 2.激光共聚焦显微镜观察：用移液器小心取出孔内的肿瘤球，用 PBS 清洗 3 遍 后，采用 4%多聚甲醛固定，并用 Hoechst 33258 对细胞核进行染色，PBS 清洗 3 遍后在激光共聚焦显微镜下观察（图 3）。
书山有 路
3D 多细胞肿瘤球的培养
原创 2017-04-20 医生科研助手
3D 多细胞肿瘤球是在体外应用组织培养方法使肿瘤细胞以多细胞集聚 体的形式生长成为具有三维结构的球体。 与传统的 2D 贴壁细胞培养模型相比，3D 多细胞肿瘤球可以通过模拟三维细胞网 络、细胞与基质、细胞与细胞之间的相互作用，从而更加贴近肿瘤组织中相应的 病理生理特征。 因此，3D 多细胞肿瘤球培养模型已经逐渐应用于干细胞培养和分化、癌症研究、 药物和毒性筛选及组织工程等特定应用中。
注意：由于琼脂糖溶液在室温时会凝固，因此从灭菌锅内取出琼脂糖溶液后一定 要快速转移至超净台内并迅速加入至 96 孔板中。
3D细胞培养PDF.pdf
3D多细胞肿瘤球的培养原创2017-04-20医生科研助手3D多细胞肿瘤球是在体外应用组织培养方法使肿瘤细胞以多细胞集聚体的形式生长成为具有三维结构的球体。
与传统的2D贴壁细胞培养模型相比，3D多细胞肿瘤球可以通过模拟三维细胞网络、细胞与基质、细胞与细胞之间的相互作用，从而更加贴近肿瘤组织中相应的病理生理特征。
因此，3D多细胞肿瘤球培养模型已经逐渐应用于干细胞培养和分化、癌症研究、药物和毒性筛选及组织工程等特定应用中。
虽然3D多细胞肿瘤球模型具有更显著的实体肿瘤生理相关性，但是与2D贴壁细胞培养模型相比，获得大量相对统一的3D多细胞肿瘤球模型需要一系列的培养过程和表征手段。
本文利用Liquid Overlay的制备方法，以乳腺癌细胞MDA-MB-231和MCF-7为模型制备3D多细胞肿瘤球并采用倒置显微镜、激光共聚焦显微镜和环境扫描电镜对其进行详细表征。
1实验前准备工作1.提前24 h取12 mL DMEM和RPMI 1640完全培养基（含10%FBS，下同）于50 mL 离心管内，置于4℃冰箱中预冷；2. 将分装好的Matrigel基质胶提前24 h从-20℃放入4℃，使其融化成液体状态；3. 将无菌的1 mL移液器枪头放入无菌50 mL离心管内，置-20℃冰箱预冷。
2琼脂糖包被96孔板1. 准确量取6 mL RPMI 1640培养基（或DMEM培养基）于2个10 mL的注射玻璃瓶内，加入90 mg琼脂糖，盖塞后放入80℃的水浴锅内加热溶解30 min；2. 加热结束后，将注射瓶放入灭菌锅内，115℃灭菌30 min；3. 灭菌完成后，迅速取出注射瓶放入超净台内。
将注射瓶内的琼脂糖溶液倒入无菌的加样槽中，用多通道移液器以每孔60 μL的量加入96孔板内。
注意：由于琼脂糖溶液在室温时会凝固，因此从灭菌锅内取出琼脂糖溶液后一定要快速转移至超净台内并迅速加入至96孔板中。
此外，为保证加样时琼脂糖不冷却，需要同时灭菌加样槽和100 μL的移液器枪头。
3d细胞球培养方法
3d细胞球培养方法## 3D Cell Spheroid Culture Methods.In 3D cell spheroid culture, cells are cultured in a three-dimensional environment that more closely mimics the in vivo microenvironment. This is in contrast totraditional 2D cell culture, where cells are grown on a flat surface. 3D cell spheroids can be used to study a variety of cellular processes, including cell-cell interactions, cell migration, and cell differentiation.There are a number of different methods for generating 3D cell spheroids. One common method is the hanging drop method. In this method, cells are suspended in a culture medium and then placed in a hanging drop on the lid of a petri dish. The drops are allowed to incubate for a period of time, during which the cells will aggregate and form spheroids.Another method for generating 3D cell spheroids is thespinner flask method. In this method, cells are suspendedin a culture medium and then placed in a spinner flask. The flask is rotated at a speed that is sufficient to keep the cells in suspension, but not so fast that the cells are damaged. The cells will aggregate and form spheroids over time.Once 3D cell spheroids have been generated, they can be cultured in a variety of ways. One common method is to culture the spheroids in suspension. In this method, the spheroids are maintained in a culture medium that is agitated to keep them in suspension. Another method is to culture the spheroids on a surface. In this method, the spheroids are attached to a substrate, such as a petri dish or a microcarrier.3D cell spheroid culture has a number of advantages over traditional 2D cell culture. First, 3D cell spheroids more closely mimic the in vivo microenvironment. This is important because the microenvironment can have a significant impact on cell behavior. Second, 3D cell spheroids allow for the study of cell-cell interactions. In2D culture, cells are only able to interact with cells that are directly adjacent to them. In 3D culture, cells are able to interact with cells from all sides. Third, 3D cell spheroids can be used to study cell migration. In 2D culture, cells can only migrate on a flat surface. In 3D culture, cells can migrate in all directions. Finally, 3D cell spheroids can be used to study cell differentiation. In 2D culture, cells are often unable to differentiate into mature cell types. In 3D culture, cells are more likely to differentiate into mature cell types.3D cell spheroid culture is a powerful tool for studying cell biology. This method can be used to study a variety of cellular processes, including cell-cell interactions, cell migration, and cell differentiation. 3D cell spheroid culture is also a promising tool for drug discovery and regenerative medicine.## 3D 细胞球培养方法。
3D细胞培养和试验系统
简介哺乳动物细胞培养作为一个无价的细胞生物学工具已有几十年。
生长在扁平和僵硬的二维(2D)底物(如聚苯乙烯或玻璃)上的单层贴壁细胞已成为传统细胞培养系统的中流砥柱。
二维细胞培养的研究在扩宽我们对发育生物学、组织形态发生、疾病机制、药物发现、大批量蛋白生产、组织工程和再生医学方面的知识方面扮演了至关重要的角色。
与此同时，随着2D培养系统而来的缺陷也显现了出来，尤其是其不能模拟体内环境以提供生理相关的数据。
在体内，几乎所有组织的细胞都处在由一个复杂三维(3D)结构的细胞外基质(ECM)中，它们和相邻的细胞通过生化和机械关系产生着相互作用1。
细胞-细胞和细胞-细胞外基质间的相互作用构建成了一个用于维持组织特异性和稳态的3D通讯网络2。
细胞生命周期的关键事件通过受到周边细胞微环境的控制3。
2D培养试验中，细胞无法实现类似于体内的结构组织和连接，这使得细胞形态、存活力、增值能力、分化、基因和蛋白表达、对刺激的反应、药物代谢及一般的细胞功能受到了限制或减弱。
2D 培养的限制对临床前基于细胞的药物和毒性筛选试验的可预测性造成了影响。
超过90&的药物通过了体外临床前研究，却在随后的临床实验中未能达到预期的疗效或安全范围4。
癌症药物的失败率更高5, 12，因为2D培养系统常常无法构建有效的肿瘤生物学模型6,7。
而且，在使用2D模型进行临床前药物研发时，生物利用率和毒物学的研究严重依赖于动物模型的使用。
这一高失败率表明动物模型可能并不适用和/或并非人用治疗方案安全性评测的代表4,16,17。
为了克服这些短板，大量的3D细胞培养模型在近20年被开发了出来。
由于3D模型显著促进了癌生物学、组织工程和再生医学的研究，又得到了进一步的发展。
为此，细胞生物学家们、材料科学家们、生物医学工程师们以及其他开发更有用的模拟体内环境的3D模型来缩短2D培养与活体组织间差距的人，携手参与了多学科的研究。
越来越多的证据表明，3D培养建立起来的生理细胞-细胞与细胞-细胞外基质相互作用可以模拟天然组织的特异性，并且比传统2D培养的生理相关性更强8-10。
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3D多细胞肿瘤球的培养
原创2017-04-20医生科研助手
3D多细胞肿瘤球是在体外应用组织培养方法使肿瘤细胞以多细胞集聚体的形式生长成为具有三维结构的球体。
与传统的2D贴壁细胞培养模型相比，3D多细胞肿瘤球可以通过模拟三维细胞网络、细胞与基质、细胞与细胞之间的相互作用，从而更加贴近肿瘤组织中相应的病理生理特征。
因此，3D多细胞肿瘤球培养模型已经逐渐应用于干细胞培养和分化、癌症研究、药物和毒性筛选及组织工程等特定应用中。
虽然3D多细胞肿瘤球模型具有更显著的实体肿瘤生理相关性，但是与2D贴壁细胞培养模型相比，获得大量相对统一的3D多细胞肿瘤球模型需要一系列的培养过程和表征手段。
本文利用Liquid Overlay的制备方法，以乳腺癌细胞MDA-MB-231和MCF-7为模型制备3D多细胞肿瘤球并采用倒置显微镜、激光共聚焦显微镜和环境扫描电镜对其进行详细表征。
1
实验前准备工作
1.提前24 h取12 mL DMEM和RPMI 1640完全培养基（含10%FBS，下同）于50 mL离心管内，置于4℃冰箱中预冷；
2. 将分装好的Matrigel基质胶提前24 h从-20℃放入4℃，使其融化成液体状态；
3. 将无菌的1 mL移液器枪头放入无菌50 mL离心管内，置-20℃冰箱预冷。
2
琼脂糖包被96孔板
1. 准确量取6 mL RPMI 1640培养基（或DMEM培养基）于2个10 mL的注射玻璃瓶内，加入90 mg琼脂糖，盖塞后放入80℃的水浴锅内加热溶解30 min；
2. 加热结束后，将注射瓶放入灭菌锅内，115℃灭菌30 min；
3. 灭菌完成后，迅速取出注射瓶放入超净台内。
将注射瓶内的琼脂糖溶液倒入无菌的加样槽中，用多通道移液器以每孔60 μL的量加入96孔板内。
注意：由于琼脂糖溶液在室温时会凝固，因此从灭菌锅内取出琼脂糖溶液后一定要快速转移至超净台内并迅速加入至96孔板中。
此外，为保证加样时琼脂糖不冷却，需要同时灭菌加样槽和100 μL的移液器枪头。
4. 加入完成后，96孔板要保持水平约30 min使孔内的琼脂糖凝固。
3
配置含Matrigel基质胶细胞悬液
1. 取对数生长期的MDA-MB-231细胞（或MCF-7细胞），胰蛋白酶消化后进行细胞计数，用RPMI 1640完全培养基（或DMEM完全培养基）将细胞悬液浓度调整至
2.0×105cells/mL，备用。
2. 将盛满碎冰的烧杯喷完酒精后放入超净台内，将RPMI 1640完全培养基以及解冻的Matrigel基质胶从冰箱内取出置于冰上。
注意：由于Matrigel基质胶在室温下溶液凝固，因此在操作过程中一定要保持低温。
3. 将预冷的移液器枪头取出放置于超净台内。
根据计算量（2.5%，v/v）用移液器将300 μL Matrigel基质胶加入到12 mL RPMI 1640完全培养基内，迅速混匀。
注意：由于Matrigel基质胶在室温下溶液凝固，因此使用的移液器枪头也需要预冷。
4. 加入步骤1中的细胞悬液（约600 μL），使细胞浓度为10000 cells/mL，迅速混匀，备用；
4
将细胞悬液铺入琼脂糖包被的96孔板
1. 将上述步骤4中配置好的含有Matrigel基质胶的细胞悬液放入加样槽内，用多通道移液器吸取200 μL加入到包被琼脂糖的96孔板内。
2. 采用低温离心机进行离心，离心条件为4℃，1000×g，10 min。
注意：离心96孔板时为保持无菌，将96孔板的周围用封口膜封住。
3.离心完成后，取出96孔板，摘下封口膜，喷洒酒精后放入培养箱内培养。
整个培养流程如图1所示。
4. 在培养的第3、5和7天，更换孔内的100 μL培养基并采用倒置显微镜观察肿瘤球的形态。
5.若要采用3D多细胞肿瘤球进行药物试验，在培养7天后，用移液器取出孔内的100 μL培养基，加入100 μL给药溶液，然后置于培养箱内培养并定期采用倒置显微镜观察肿瘤球的生长状况（图2）。
5
3D多细胞肿瘤球的表征
1. 倒置显微镜观察3D多细胞肿瘤球形态：直接将96孔板置于倒置显微镜下观察即可。
2. 激光共聚焦显微镜观察：用移液器小心取出孔内的肿瘤球，用PBS清洗3遍后，采用4%多聚甲醛固定，并用Hoechst 33258对细胞核进行染色，PBS清洗3遍后在激光共聚焦显微镜下观察（图3）。
3.环境扫描电镜观察：用移液器小心取出孔内的肿瘤球，用PBS清洗3遍后，固定干燥后进行环境扫描电镜观察（图4）。
图4。