Effects of Static and Dynamic Culture Conditions

Effects of Static and Dynamic Culture Conditions create from raw material engineering has been investigating the goodties of sustains and st exclusively in allph oneness gloss fountains for better cadre union, viability and proliferation. This say equates the two cubicle ending particularises Static and spinster flaskfulful / impulsive electric booth figures over a period of 7 solar sidereal sidereal mean solar solar daytimes on polyglycolyic acid. The scaffolds were statistically shed by mouse cutaneal 3T3 fibroblast in smooth refining order acting and on other hand seeded scaffolds were transferred to spinster flask at approx.60 rpm in fighting(a) refining method. substantial improvement in carrel viability was not spyd in twain the conditions after 7 eld of culturing. The stalls adhesion successfully took place and express cytoskeleton -actin in both the methods save achieving uttermost distribution of electric prison jail cadres on th e scaffold in impulsive method. This count reports that nonoperational ending method could nurture summation in stall come approximately six propagation more than than after 7 years of refining i.e. from 1.2 x 10 (0.1610) cubicles to 6.3 x10 (110) cells. Surprisingly, instead of enhancing the growth of 3T3 fibroblast cells in kinetic condition, they seems to be credibly at a lower placegoing cell death/loss as reported by alamar highish, hoechst desoxyribonucleic acid hitchs, toludine puritanic and western pip. Overall, nonmoving condition favoured the cell adhesion, proliferation and -actin mien step by step with years and dod better reproducible selective information comp atomic number 18d to high-power condition. The techniques relate in energizingal finis method needs to be more cautiously investigated and improved further to draw a strong conclusion.The aim of the study is to implement the principles of fundamental techniques in wind engineering in tillage method on the three dimensional polyglycolic acid (PGA) scaffolds seeded with 3T3 fibroblast. To comp be and contrast the nucleuss on cells in spinner flask or dynamic burnish condition method with the atmospherics finishing condition method by observing and analysing on factors like cell adhesion, distribution, proliferation, viability and expression of cytoskeleton after culturing in the same system for 7 days victimization alamar gruesome, hoechst 33258 desoxyribonucleic acid assays, toludine blue dye and western place analysis.Tissue engineering is a multi dish aerialiplinary field which aims in developing new approaches for functional substitutes applic fitting in restoration of damaged or injured tissues. These substitutes are compound constructs of living cells, bioactive molecules and three dimensional porous scaffolds, which supports cell fond regard, proliferation and differentiation. Therefore, its main neutral to carry push through in therapy is to form a living tissue from small population of mammalian cells. For this, the ideal tissue engineering strategy so far has remained to develop tissue by seeding the specific population of cells on third-dimensional constructs which not save provides a structural support to cell mass but as well back end usefully influence cell adherence, growth and differentiation either by incorporation of adhesion molecules or controlled release on bioactive molecules from the scaffold. After seeding of cells onto the 3-D scaffolds construct, the cells starts proliferating which results in deposition of extracellular matrix components and biodegradation of scaffolds. The latter makes the porous construct of scaffold more lusty 3-D.Several other factors disturb the 3D tissue growth including scaffold stick out, seeding method and the market-gardening condition methods. Studies acquit reported that mellow gradation of cell attachment to biocompatible and biodegradable particles, whi le negateing aggregate arrangement can be achieved victimization poly co-glycolic acid (PGA) scaffold of 50- hundredmm spherical size fabricate by electro- gyrate technique. This method provides undeviating, reproducible and wellspring-characterized PGA scaffold. The protrude chemistry of the scaffold helps to check out the particle size, shape, morphology and distribution. Depending on the experiments, surface modifications are performed like shaping of poly l-lactide-coglycolide (PLGA) via ring opening polymerisation and fibronectin coating to scaffolds. However, it is not the part of timeworn protocol. Depending on the size, the required cell parsimony for maximum attachment may differ to applyed optimal cell attachment.The seeding is usually make victimisation the cell suspension of a particular seeding immersion which allows for maximum dispersion of cells and well integration into the pores of the scaffolds. however for therapeutic purposes, however, this stra tegy is not sufficient luxuriant to result in an overall improvement in conditions due to dreadful tissue damages. This can be overcome merely by achieving comparatively high degree of cell attachment to the micro-particle. Several factors and parameters influence the cell adhesion like the curvature of the particles, the particle material, the electro nonoperational charge of the particles, the surface motif of the particles, the interaction between cell and particles, the number of cells in the tissue flori glossiness and type of cell culture method implemented. It is likewise beta to obtain homogenous cell adhesion to the scaffolds and avoiding clumping which will lead to the constitution of cell-particle aggregates. This will prevent cells from attach uptake of nutrient from the media and hinder their attendant growth.The mammalian cells are usually cultured in electro smooth or bioreactors condition. Here in this study, spinner flask system is employed which is in a ddition a kind of bioreactor as it provides the 3D environment. It is a flask provided with magnetised rod which keeps rotating constantly at specified speed. The nature of growing cells requires much(preno bital) dynamic condition to mimic the environment similar in perspective the consistency which gives sufficient nutrient supply, flagellate exchange, enhances ECM and gap junction formation, and cell-cell interaction. close importantly it besides helps honour the cells differentiated in 3D which is inevitable for tissue formation. This characteristic is not maintained by stable culture method. Hence, many 3D culture methods have been developed such as perfusion chambers, rotary vessels and commercial perf employ bioreactors with improved capacity for mass transport of nutrients and waste product. They help in formation of relatively good quality of tissue by more enhanced cell differentiation and similarly maintaining in that state. The static culture method used in thi s study, tissue culture plastic with seeded scaffolds remains untouched in the incubator. But with static culture, alternative shaking on a shaker and resting can overly be employed to provide better supply of nutrient through and through media.The attachment characteristic of ECM proteins such as laminin, will also depend upon the cell type used. There are particular conditions undeniable to be honed with each cell type. Most of the tissue engineering experiments uses 3T3 fibroblast only to optimise the cell culture condition where there is optimum cell adhesion is obtained before apply the actual stem cell of interest. This is because, 3T3 fibroblast are known to easily attach to any surfaces due to presence of the high niggardliness of integrins on their cell surface. This will not only enhance the cell attachment but will also give maximum possible interaction with the particle. Cells that have spread on the particles exhibit a clear halo of cytoplasm surrounding their nuc leus after the rearrangement of their actin skeleton. The attachment and spreading of cells to a substrate surface is often seen as a basic characteristic, but is, in fact, the initial process that subsequently influences and regulates cell growth, survival, migration and differentiation. In addition, cell-to-substrate interaction, mediated by integrins, also influence cell conduct and signalling pathways leading to modifications in upstream and/or downstream cellular activity. Thus, a desirable substrate should allow sufficient and optimal cell attachment and spreading characteristics to occur. The 3T3 fibroblast media is used in which DMEM supplemented with 10% FCS enhances the cell attachment as the blood serum is highly protein rich and therefore, helps in cell in adhesion by supplying the ECM-proteins as well as nourishing them. Hence, the serum conditioning step is of critical importance in maintaining cells health and attachment in the culture.Materials and MethodsScaffold p reparation and serum conditioningPGA FELT Scaffolds disc of 2mm x 10mm and 45mg/cc (TE005-50-10) was provided by Smith and Nephew research group, University of Nottingham. These non-culture scaffolds were hence hard-boiled in 24 well tissue culture plastics (TCP) carapaces with 3T3 fibroblast media containing 500ml DMEM (Sigma G7513) supplemented with 10% FCS, 2mM L-glutamine and 1% AB/AM (Sigma A5955).All the scaffolds were statistically seeded on day 1 using non-culture treated well headquarterss to encourage the cells of mouse dermal 3T3 fibroblast to adhere to the scaffolds at seeding density of 1x 10cells/ml. 3T3 fibroblast cell suspension was added in TCP plates for all shew and no cells in the blanks. The plates are thus incubated overnight at 37C, 5%CO in manner. The be cell suspension was indeed again resuspended in warm media to achieve 4 x 10 cells/ml cell density and was stored at -20C till day 7 for Hoechst analysis.3T3 fibroblast cells were used to seed the s caffolds to recover the cell viability, cell proliferation and -actin expression on day 1, when the cell culture condition was maintained static and day 7, after applying the two cell culture conditions (static dynamic) and maintaining for 7 days.Static cultureIn static culture condition, the seeded and non-seeded (blanks) scaffolds were unbroken in 1ml of warm 3T3 fibroblast media per well. These five culture plates were kept in incubator and cultured for 7 days at 37C, 5%CO in air.Spinner flask culture dickens separate spinner flask filled with 50ml warm media each was used for seeded scaffolds and non-seeded (blanks) scaffolds. These flasks were kept in incubator by loosening the side arms and sufficeting the magnetic stirrer approximately at 60rpm and cultured for 7 days at 37C, 5%CO in air.After following 7 days for culture conditions, the construct was then sacrificed for alamar blue, toludine blue and Hoechst analysis. Also, in addition cytoskeleton analysis using wester n blot was also carried out. The assessment of two culture methods, static and dynamic was through with(p) by producing five set of readings for static condition and four set of readings for dynamic condition where the experimental analysis were conducted using three replicates for stress and blanks on day1 and day7Alamar Blue AssayStaining was done using 10% alamar blue containing 1ml alamar blue (Serotec BUF012B) and 9ml HBSS without phenol red (Sigma H1387). The stain was kept in unlighted at 37C. The scaffold was transferred from seeding and culture conditions to new 24-TCP non-cultured plate with 1ml warm alamar blue after washout three times with PBS. The plates were then incubated at 37C, 5%CO for 1hr. The aliquots of 3 x 100l of alamar blue were transferred to 3 wells of 96 microtitre well plate including the blanks to measure fluorescence using plate reader (Ex530nm/Em590nm). The excess of alamar blue dissolver was aspirated and washed with 1ml sterile PBS.Toludine Blu e StainingScaffold for toludine blue staining was transferred to new non-culture treated 24well TCP plate and was treated with 1ml ice ice-cold 95% (v/v) methanol in dHO for 5 mins after rinse 3 times with 1ml warm PBS. Then fixative was discarded and scaffold was allowed to air dry at RT followed by treatment with 1ml aqueous 0.1% (w/v) toludine blue (Fisher chemicals BPE107-10) for 5 mins. The scaffold was again allowed to air dry at RT.Papain Digest and Hoechst 33258 DNA AssayThe aliquot of cell guessing (4 x 10 cells) active on day1 was treated with 1ml of papain answer (1.06mg/ml, pH 6.5) (Sigma P4762) followed by overnight brooding in waterbath at 60C. The serial dilutions of the papain digested cell nip using hydrolysed papain solution as diluent was prepared for 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10 cells. In the Hoechst 33258 DNA assay, the hydrolysed papain solution was used as blank. 5l of each aliquots + 70 l Hoechst dilution buffer was added i n triplicates in dismal 96-well plate including the blank. In each well, 100 l Hoechst 33258 working solution (Sigma S6639) was also added and fluorescence was measured using plate reader (Ex 360nm/Em 460 nm) westbound Blot100l cold RIPA buffer (Sigma R0278) was added to the cell pellet (4 x 10 cells) and the seeded scaffolds in eppendorf from day 1 and was kept on ice for 20 mins while vortexing every 5 mins. The cells were then snap freezed by placing it on dry ice for 1 min then 1-3 min at RT. The cells are resuspended by grating and spinning for 30 mins. The supernatant was used for western blot. 10l of molecular weight marker and each sample were loaded onto SDS polyacrylamide gel. The electrophoresis was carried out for 90 mins at 125V. After SDS-page electrophoresis, the filter paper, nitrocellulose and sponge were sop in transfer buffer (Invitrogen NP0006) with 20% (v/v) methanol. The assembled western blot armoured combat vehicle was run for 1 hr at 25V. The immune-dete ction of protein -actin was performed using primary antibody anti-mouse -actin (Sigma A2006) and collateral antibody anti-mouse horse radish peroxidises (HRP) (Invitrogen G21234).Statistical analysisAll the selective information obtained was calculate using MS-Excel spreadsheet and statistic Independent t-test and mated t-test analysis was performed using SPSS software.Results and Discussion sound structure of 3T3 fibroblast cellsThe cell of mouse dermal 3T3 fibroblast was obtained from T180 flask by trypsin digest method is projectn in interpret1. The flask was confluent enough (80%) and morphology of the cells seems to be inbuilt and healthy. No sign of contamination was observed introductory to seeding procedure. The morphology of 3T3 fibroblast cells are of flat and spindled shape. These cells form a well-characterised and established mesh like merged networks. This property of fibroblast cells make them ideal for cell attachment as they show anchorage property due to pre sence of integrins in ECM. Hence, using this cell type achieving maximum cell adhesion onto the scaffolds becomes ideal for this experiment.Effect on Cell viability in static and dynamic conditionsThe alamar blue assay was performed on the static and dynamic culture condition to observe its effect on 3T3 fibroblast cell viability is shown in figure 2a and 2b. The culture method employed aims to maintain or increase the cell viability when cultured for sevener days. Under static condition (Fig 2a), only 1 group out of five showed world-shaking increase in fluorescence whereas other two groups showed more or little no change in their fluorescence produced from day 1 to day 7. Also, on contrary two groups showed authoritative decrease in fluorescence on day 7 (Fig 2a). Hence, variable of results were obtained between groups. On the other hand, under dynamic condition, the cell produced more fluorescence on day 7 compared to day1 expect for one group. Therefore on an average, when m ean of the static absorbance reading was taken, it showed that there is square decrease in fluorescence (fig 2b). But in dynamic method, the increase in fluorescence day 7 (Fig 2b) was not significant enough. The 3-D construct of PGA scaffold provides with an environment to the cells where they remain viable in culture for several weeks. Moreover, they should successfully increase the cell viability after some days. However, our study reported that the cell viability decreased tremendously in cell seeded PGA scaffolds in static cell culture condition whereas the dynamic cell culture method was able to increase the cell viability over 7 days of culture. So, when analyse the two culture methods statistically showed difference in their overall effect on the viability of 3T3 fibroblast cells where dynamic condition is more but not effective enough. So, static condition did not improve the cell viability more than dynamic culture method.Effect on Cell distribution in PGA scaffoldsThe t hree-dimensional PGA scaffolds constructs enables the fibroblastic cells to adhere and to evenly distribute throughout the porous structure. To assess the ordered 3T3 fibroblast cell distribution in two different culture conditions, toludine blue staining was carried out on day 1 and day 7 on both conditions is shown in fig 3. Toludine blue stains cell dark blue inside the 3-D construct. As observed in static condition, on day 1 the cells were successfully seeded onto the scaffold but compared to day 7 the cells are not evenly distributed throughout the scaffold. Also, the scaffolds were efficiently seeded on day1 under dynamic condition as the figure 3c shows cells stained with toludine blue. Surprisingly, on day 7 (Fig 3d), the scaffolds shows no cells at all. This means, that the 3T3 fibroblast cells under dynamic condition was last lost(p) or died. The spinner flask culture system might have loose the cells by day 7 due to paltry adhesion or vigorous rotary motion. The cel l seeded on day 1 was too low or error in carryout the technique. But this was observed with all the spinner flask condition system, where the success was 2 out of 4 groups (Supplementary data 3). However, this observation is more of debate because no other factors expect the condition itself could affect cell distribution as uniform distribution was achieved in all the five static condition (supplementary data 3) which used the same scaffolds and cell type.Effect on Cell proliferation in static and dynamic conditions3T3 fibroblast was culture over 7 days in both conditions to also observe its effect on the cell proliferation are shown in figure 4 (a, b, c d). The model rationalise obtained with known cell density for both static and dynamic of all the groups (fig 4a 4b) showed increase in cell density with increase in the fluorescence. The unknown cell density of the cells from these two culture methods on day 1 and day 7 was calculated and ensnare that 2 out 4 groups from dy namic conditions had no cells in the culture on day 7. Therefore, only other two groups were considered to evaluate the cell number on day 1 and day 7. There was significant difference in cell density over 7 days of culture in static method (n=5)(fig 4c) and on contrast there was no significant difference in cell density in dynamic method (n=2)(fig 4c). Almost all the groups showed cell density on day 1 around 1 x10 cells/ml which was the actual cell density seeded on day 1 (supplementary data 4). This shows that seeding performed on scaffold achieved effective adhesion of all the cells present.The mean cell number from 1.2 x 10 ( 0.16 x 10) cells on day 1 increased to 6.3 x 10 ( 1 x 10) cells on day 7 under static culture method (fig 4d). On the other hand, dynamic culture methods showed hardly any change in cell number over 7 days of culture i.e. 2.0 x 10 ( 0.92 x 10) cells on day 1 to 2.5 x 10 ( 1.96 x 10) cells on day 7 (fig 4d). antecedent studies have reported using other cel l types that they start proliferating within 24 hrs after seeding cells on scaffolds employing dynamic culture methods. Contradicting this, our results have shown that dynamic had really poor effect on cell proliferation. Moreover, 3T3 fibroblast cells were undergoing death during seven days of culture. Whereas, static culture method shows drastic increment in the cell number and thus supporting 3T3 fibroblast cell proliferation efficiently. The scaffolds used for alamar blue assay on day 1 were used for Hoechst DNA assay with same after washing step (same for day 7 scaffolds). The washing might have been too vigorous which resulted in cell loss. It could also be possible that cells are being aspirated off from the culture which gave poor or no cell proliferation. It should be also taken into cover that the success rate with dynamic culture method on cell proliferation was null out of 4 demonstrations.Expression of CytoskeletonFor the analysis of expression of cytoskeleton -actin o n 3T3 fibroblast in two different conditions was done by western blot as shown in figure 5. The cell pellet of density 4 x10 cells/ml was loaded against the cells obtained on day1 and day 7 from static and spinner flask culture method. The density of -actin obtained from the cell pellet was maximum. The amount of -actin detected on day 1 was lower than day 7 in static culture condition. It was the opposite scenario with spinner flak method where day 7 had minimum amount of -actin compared to day1. In some cases of spinner flask method -actin was not even detected on day 7 (supplementary data 5). Hence, comparatively the expression of -actin was higher in static culture method. Perhaps, it could be because the cell could not proliferate much as expected. Also, the culture didnt have enough cells left to express -actin on western blot. The formation of ECM cytoskeleton was not shown to be supported by spinner flask method.Conclusion and future workThe tissue engineering scaffolds con structs have been shown more effective on cells containing serum in spinner flask/dynamic culture method rather than in static culture method. But from our data, it shows that dynamic condition only favoured cell adhesion and distribution. It was also able to produce a small increment in cell viability dissimilar static culture method. Contradicting the other data, cells were virtually not detected on day 7 and so is the expression of -actin. Not only this, all the 4 demonstration failed to show that cell growth can be in effect supported in dynamic culture method. Three seeded scaffolds were kept in spinner flask together, where there is increased possibility for it to come in contact with each other. Cells may get marooned from the scaffolds as it might be loosely adhere to the scaffold. The continuous rotation of magnetic rod in the flask circulates the media to provide nutrients to cells more effectively then static. Despite of this fact, the cells were either undergoing cell death or dislodged from the scaffolds or may be aspirated off from the culture.The static culture method have been effective in 3T3 fibroblast adhesion on the construct after seeding and eventually could improve tremendous cell growth by showing increase in cell proliferation over a period of seven days in culture. However, better distribution and increased -actin expression could only be achieved by the static culture method after 7 days as the cells proliferated more. Moreover, the success rate for this method was more compared to dynamic and produced more reliable and reproducible data. Hence, it can be concluded that static culture method supported cell growth better then the dynamic culture method.It would be interesting to investigate the technique involved in dynamic culture method more carefully to produce reliable data where it could be compared with the static method to give better cause of the environment cells require to grow in artificial ECM-like structure and cultur e media. Since, within the body the cells are continuously under the force by blood flow in 3D environment, it would be useful to withhold cell culture growth better in dynamic condition with enhanced technique. It is strongly recommended to carry out further research in this area to conclude spinner flask methods effect on 3T3-fibroblast cells with more reliable data.EvaluationThe practical seance assessed my learning in the techniques and concepts involved in tissue engineering. The demonstration on different techniques to prepare scaffolds assessed my taking into custody better and was helpful to apply same in this practical session by evaluating the different parameters that can be influenced by the scaffold design alone. As earlier discussed troubleshoot, implementing the technique given in protocol helped to produce the good replicates and contamination free-blanks and controls. While working in the hood with the partner, things were discussed prior to carrying out the expe riment and working space was kept ready which helped in managing the use of same equipments, solution and incubation time effectively to avoid any source of contamination. Also, the exchange of results and data between several groups also led to the exchange of ideas and different cause for their results. However, the exact reason for spinner flask method to not work out is still not clear as all the groups got same reading where cells were present onto the scaffolds during alamar blue assay on day1 and day 7 but eventually lost when subsequent assays were done for same day. Overall, the difference between the effects of two culture method was evaluated.AcknowledgementThe efforts put in by the Paula Ellis is admit was carryout the change of media and taking care for the samples throughout the practical session and also during weekends. Also, Dr. Felicity Rose for giving the guidance and helping with doubt regarding the techniques and protocol. The images and data taken from all othe r groups are declare for sharing their data used in this report. The effort of the group member is also acknowledged for managing with the working protocol load effectively. rendersFigure1. 3T3 fibroblast cells in T180 flask (10X). The image shows morphology of 3T3 fibroblast prior the trypsin digest followed by static seeding. The image was taken using Nikon (Scale bar 80m)Figure 2a. Alamar blue assay for all static (n=5) and for dynamic (n=4) culture methods on day1 and day 7. The graph shows fluorescence detected SD for both the culture condition. The absorbance value of non-seeded scaffold (control, Ac) was subtracted from the absorbance value obtained for seeded scaffold (As) to optimise the calculated fluorescence i.e. As-Ac. This was done for all the static and dynamic culture methods data. The statistical analysis paired t-test was at 95% significance level was done using SPSS. The calculated data is provided in the supplementary data.Figure 2b. Alamar blue assay of static and dynamic condition on day 1 and day 7. The mean of all the values on day 1 and day 7 for static (n=5) as well as dynamic (n=4) was done. The graphs shows the mean of absorbance (O.D) SD. The statistical analysis was performed using paired t-test and independent t-test at 95% significance level.DAY 1 DAY 7Figure 3. Toludine blue assay. The toludine blue staining was performed on static culture condition on day 1 (a) and on day 7 (b). besides for dynamic culture condition on day 1 (c) and day 7 (d) was carried out. In (a) and (b) there is darker background staining but (c) shows proper stained 3T3 fibroblast cells. No cells staining can be detected in (d) (Scale bar 100m).Figure 4a. Standard curve for all static condition using Hoechst 33258 DNA assay. The standard curve was plotted using the known concentration 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10 (blank) of 3T3 fibroblast cells against the absorbance obtained. The blank was subtracted from the test readin g to standardise the graph. The graph was produced using MS-Excel, to obtain the linear reverting and linear equation for each group to calculate the cell density in static culture condition.Figure 4b. Standard curve for only two dynamic condition using Hoechst 33258 DNA assay. The standard curve was plotted using the known concentration of 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10 (blank) of 3T3 fibroblast cells against the absorbance obtained. The blank was subtracted from the test reading to standardise the graph. The graph was produced using MS-Excel, to obtain the linear regression and linear equation to calculate the cell density in dynamic culture condition on day 1 and day 7.Figure 4 c. Hoechst 33258 DNA assay was carried out on all static (n=5) and dynamic (n=2) culture condition on day 1 and day 7. The cell density was calculated using the standard curve for its own respective group. The graph shows cell density (x 10 cells/ml) SD for static and dynamic condition. The slowness was performed on excel-sheet and statistical analysis of paired t-test was done using SPSS.Figure 4 d. Hoechst 33258 DNA assay. The unknown cell density calculated from standard curve was averaged (mean) for static (n=5) and dynamic (n=2) culture methods. The graph shows cell density (x 10 cells/ml) SD for static and dynamic condition. The calculation was performed on excel-sheet and statistical analysis of paired t-test and independent t-test was done were appropriate using SPSS.Figure 5. Western blot analysis of 3T3 fibroblast cell from static and dynamic on day 1 and day 7. The expression of -actin in both culture methods are analysed using the rainbow marker and compared with the actual pellet of 3T3 fibroblast to cells extracted from two different culture methods on different days.