Progress of project for week 3:

Conducted experiments to figure out the best mixtures of gelatin and alginate for producing a gel that is able to simulate the collagen in our body for containing and adhering of fibroblasts. Assembled the prototype spray mechanism for the spraying of the keratinocytes. Subsequently, over the next 3 days, measurements of the weight loss and degradation of the gels are recorded to test our hypothesis if the following two factors would affect the degradation of the gels:

1. Low temperature vs Room temperature
2. Wet vs Dry conditions

Results:

From our results, it is clear that in order to reduce the degradation of the gels, the gels would have to be kept at low temperatures and soaked in water. However, more experiments would have to be conducted with fibroblasts in order to find out the optimum mixture of gelatin and alginate that would yield the highest survivability of the fibroblasts and also whether the temperatures and wet/dry conditions would affect the survivability of the fibroblasts.

 

 

Progress of project for week 4:

Midpoint check-in 1

Presented on project preliminary ideas and progress of project. Received comments on focusing the project around improving the consistency and precision of the skin making process rather than on speed as the machine need not necessarily be more efficient as compared to humans in making the skin.

Safety training at LKC

Project officer at LKC brought us around the laboratory and briefed us on the things to take note of when using the lab. Also managed to get opinions from him and the other people at the laboratory on the mediums to be used for the fibroblasts and keratinocytes. Cell-lines to be used and the method of incorporation of the fibroblasts into the fibroblast gel layer requires further discussion with project supervisor Professor Andrew Tan Nguan Soon.

Meeting with Mr Tony Gan Eng Swee (Making and Tinkering advisor)

Mr Gan recommended the use of a peristaltic pump with a stepper motor for the delivery of the gel layer and the keratinocytes. However, upon further discussion, he also thinks that the team’s original idea of using a syringe instead of a peristaltic pump might also work. For now, the team has decided to try out both mechanisms to see which one works better.

Team Meeting

From the meeting, the team has decided to purchase a peristaltic pump with a stepper motor, a spray nozzle (similar to those used in sprinklers for keratinocytes), icing piping syringe nozzles (for fibroblast gel layer) and a syringe.

 

 

Progress of project for week 5:

3D printer assembly

Assembled the Ender 3D printer to be used for the printing of the skin. Also purchased another peristaltic pump with a higher flow rate and pressure.

Meeting with Associate Professor Tan Nguan Soon

Confirmed the feasibility of growing fibroblasts within the gel layer and the method to check for fibroblasts and keratinocytes survival. Also decided on the method of preparation of the gel layer with the fibroblasts and the keratinocytes. Have to take note of that the calcium ion concentration cannot be too high or else keratinocytes would differentiate too early and ceased to proliferate, stopping the skin growth.

Cell culture training

Got trained on cell culturing techniques and protocols and was given a batch of fibroblasts and keratinocytes to take care of. Preparation of cell culture medium was also taught to us. Both the medium and the cells will be used for our future experiments.

 

Gel layer experiment

Prepared a gel layer from alginate and gelatin under sterile conditions. Gel layer was incubated over two days and then put under the microscope. It was found that 1 out of the 2 alginate-gelatin layers are contaminated. A longer UV duration was suggested by Prof Tan for the gel layers in order to ensure that they are properly sterilised. Dry gelatin, alginate and calcium chloride in their powdered form are place in the biosafety cabinet and placed under UV for 3 hours. These will be used in the following week for the making of the gel layer. It was also found that alginate is extremely viscous and hard to dissolve completely in DMEM. Further changes to the concentrations of each component; alginate, gelatin, calcium chloride and DMEM will be made for the experiments in the following week.

 

 

Progress of project for week 6:

Gel layer experiment

Prepared gel layers with different proportions of alginate and gelatin (A:G, 30:70, 40:60, 50:50) each with one gel soaked in DMEM and one that is dry to investigate the degradation of the gel layers without the DMEM. 2% w/v alginate is made by dissolving 4g of alginate in 100ml of MilliQ water. The alginate mixture is then microwaved in order to dissolve the clumps of alginate. The mixture is then autoclaved for 1.5 hours and 100ml of 2X DMEM is then added to the mixture to get the 2% alginate. For 10% w/v fish gelatin, it is prepared by dissolving 5g of fish gelatin in 50ml of 1X DMEM. For 3% calcium chloride, 3g of calcium chloride is dissolved in 100ml of MilliQ before being filtered using a 0.22um syringe filter. Cell density of NiH 3t3 fibroblasts from the passaging are then calculated through cell counting using a haemocytometer.  2ml of the gel mixtures (alginate+gelatin+cell suspension+DMEM) are then added to each well for the respective proportions and 1ml of 3% calcium chloride are added to each well for 15mins before being removed using the micropipette. 1ml of 1X DMEM is then added to wells that are supposed to be soaked.

Results: Fibroblasts remain spherical in shape with seemingly no changes in shape suggesting that the cells are not adhered to the gel layer. However, the gel layers managed to remain relatively solid.

Midpoint check-in 2

No new comments apart from some queries regarding our choice of nozzles for the gel layer and spray.

2nd gel layer experiment

As the fibroblasts seems to be suspended in the gel layers with no protrusions growing out of them, it was suggested that the alginate amount might be too high for the fibroblasts to change shape. Another possible reason could be that the concentration of the calcium chloride used was too high. In order to check whether the amount of alginate in the mixture is the problem, Prof Tan suggested to try with a gel layer made out of just gelatin and see if the fibroblasts changes shape before trying with alginate. Another well with gel layer of the proportion (A:G, 10:90) was also made to see if a lower alginate amount is sufficient to bring about a change in shape of the fibroblasts. As the purpose was to test if alginate amount is the problem, calcium chloride concentration is not modified and remains at 3% w/v. The gelatin concentration is also increased to 30% w/v as suggested by Prof Tan. However, the gelatin is extremely viscous at this concentration and requires a lot of heating at 65 degree celcius in a water bath before it can be used.

Results: The fibroblasts changed shape in the 100% gelatin but not in the 10:90 A:G gel layer. This suggests that the problem, as we suspected, is likely due to the use of too much alginate or calcium chloride in the making of the gel layers. Our main objective now is to moderate both the amount of alginate and calcium chloride used such that the gel layer is a solid piece and at the same time allow the fibroblasts to adhere.

Peristaltic pump tests

Using the Ender 3D printer and a micro-controller, we tested out both the peristaltic pumps that we bought with water and are able to control the flow rate using Arduino and Marlin, the micro-controller. For next week, we will be trying it out with the viscous gelatin gel layer and also write out the G code for the automated printing process.

 

 

Progress of project for Week 7:

Gel Layer experiment

The gel layers are made in the same way as before, this time using proportions of (A:G, 1:99, 2:98, 5:95). Gelatin concentration used is 30% w/v and alginate concentration used is 2% w/v. Calcium chloride concentration used this time is 1% w/v instead of 3%w/v.

Results: Fibroblasts changed shape in the wells 5:95 and 2:98. It seems like the fibroblasts are attached to the gel layer instead of the bottom of the petri dishes after consulting other members of the lab for the dry gel layers that are not soaked in medium. For the gel layers that are soaked, it appears that the gel has degraded and the cells are adhering to the bottom of the petri dishes. However, we cannot be certain about these conclusions and we came up with another idea for the gel layers.

2nd gel layer experiment

The new idea is to form an alginate layer at the bottom of the well first, before adding the gelatin layer with the fibroblasts on top which we called the “sandwich” model. 2% w/v alginate is spreaded at the bottom of the well before 3% w/v calcium chloride is added for cross-linking of the alginate to take place. Calcium chloride is then pipette out and gelatin is added on top of the alginate layer along with the fibroblasts. This should allow easy removal of the layered construct with the alginate forming a scaffold at the bottom of the well, while at the same time, providing the gelatin required for the fibroblasts to adhere to and change shape.  We also repeated the previous experiment using 5% w/v alginate this time instead of 2% w/v alginate and different proportions (A;G, 2:98, 3:97, 4:96) as we find that our gel layers do not remain solid after a while and may require higher alginate concentration.Gel layers of the proportions (A:G, 2:98, 3:97, 4:96) were also made in a similar way as described in the previous experiments and were  used for the seeding of keratinocytes in order to find out if they could grow on these gel layers. If both the fibroblasts and keratinocytes are capable of growing on any of these proportions (A;G, 2:98, 3:97, 4:96), this would mean that our experiments are highly successful and the only thing now would be to grow both the fibroblasts and keratinocytes on the successful gel layer and see if they could grow into the skin that we wanted.

Results: There are no change in shape for both fibroblasts and keratinocytes in the gelatin+alginate gel layers. However, fibroblasts have changed shape in the “sandwich” model and we have verified that it can be peeled off easily therefore, from here onwards, our experiments will be focusing on using this “sandwich” model as summarised in Figure 10.

 

Finishing the assembly of the machine

The Marlin micro-controller was attached to the 3D printer and is able to control the printer nozzle successfully. We also tried extruding the gel layer using the stronger peristaltic pump and is able to get a quite even layer. Previous experimentations with water also suggested that the peristaltic pump is able to extrude a fixed amount of gel per step with high precision. Most importantly, we have finished the G code for the printing process and have verified that it would work in the way we wanted with the 3D printer. The only things left to do now would be to try it with the nozzles that we have gotten, make a holder for the nozzles and also try out the entire printing process with both the gel layer and the aqueous layer that would eventually contain the keratinocytes. We also decided to change the way the machine will deposit the 2nd layer. Instead of spraying, we have decided to do it via dripping as the weaker peristaltic pump meant to perform this is unable to produce enough force for spraying. Moreover, this will also maximise the number of surviving keratinocytes by reducing the shear stress caused by the spraying on these cells.

 

 

 

Progress of project for Week 8:

Gel layer experiment

As we have previously found out that the fibroblasts do change shape in our “sandwich” model, we now have to verify that this did not occur by chance. We made 6 technical replicates in total on a 6-well plate. If the fibroblasts in all the wells changed shape, this would verify that our model is indeed successful in creating a layer that could be peeled off and at the same time allow the fibroblasts to adhere to and change shape. Our next objective would be to test if the keratinocytes could also adhere to and grow on the gelatin. Prof Tan also suggested that we try collagen as well to see if the keratinocytes could grow on the collagen. If either gelatin or collagen is successful, we could simply transfer the layer with the keratinocytes on them over to the fibroblasts layer and this will complete our “sandwich” model. We made a total of 6 wells, with 3 wells containing gelatin and 3 wells containing collagen. However, due to lack of time, we only manage to seed the keratinocytes on the wells containing the gelatin and the collagen wells were left empty.

Results: After 4 days, out of the 6 wells for the fibroblasts plate, only 4 of them contained fibroblasts which have changed shape. The remaining 2 were contaminated and hence did not have fibroblasts which changed shape. However, this is sufficient to show that the fibroblast layer for the “sandwich” model is capable of allowing the fibroblasts to change shape. In our following experiments, we will need to improve on our aseptic technique while making the gel layers in order to ensure that there are no contamination to the gel layers. For the keratinocytes, it seems like they did not survive the 4 days and hence we are unable to conclude yet whether they can adhere to gelatin. Prof Tan suggested that this is likely due to the fact that the keratinocytes were exposed to air before they can grow and reach confluency and that we should redo the experiment for the keratinocytes as described below.

Gel layer experiment 2

For the keratinocytes, we decided to seed at a higher density and hence, as we do not have enough cells, we decided to only have 2 wells containing gelatin and 2 wells containing collagen. We seeded the keratinocytes on top of the layers and waited for around 3 hours for the keratinocytes to adhere to these layers before adding DMEM medium to the wells to ensure that the keratinocytes are not exposed to air while they continue to grow. However, our work here is not done. Once the keratinocytes have reach confluency, we will have to take out the DMEM medium and expose the keratinocytes to air in order to promote differentiation of the keratinocytes which will help give rise to the outer layers of the skin. For the preparation of the collagen, we dissolved collagen in 0.5M of acetic acid and then added HBSS (Hank’s Balanced Salt Solution) containing phenol red to the collagen. We then neutralise the acid using 10M and 1M NaOH using the phenol red as an indicator before pipetting them into the wells for formation of collagen layers.

Results: Both gelatin wells are contaminated. For collagen, we used collagen that we have neutralised previously a few days ago. However, collagen cannot be stored in its neutralised form and should only be stored in acidic conditions. As a result, we decided to redo both experiments for gelatin and collagen.

Mounting of stronger peristaltic pump onto the machine

We mounted the stronger peristaltic pump onto the machine using leftover aluminium frame in the MnT lab with hammer nuts. The aluminium frame was tied to the frame of the 3D-printer using cable ties while the peristaltic pump is mounted on the aluminium frame. We also printed a nozzle holder and attached it to the machine. We then tested the machine out again with our G code and it is capable of dispensing the viscous alginate. We have also integrated into our G code a command for filling up the tubings of the peristaltic pump with the alginate before the printing process. However, we have yet to test this modified G code out with the alginate and will finish this up in the following week.

 

 

 

Progress of Project for Week 9:

Gel Layer Experiment

We followed the same procedure used in the previous week for the preparation of the gelatin wells and seeded the keratinocytes on top of the gelatin layers. However, for collagen, Prof Tan suggested that in order to test the adhesion of the keratinocytes to the collagen layers, we can simply use collagen that are neutralised with PBS instead of having to add the HBSS and neutralising with NaOH. Therefore, for the preparation of the collagen, we used the collagen that we have prepared previously in 0.5M of acetic acid that have not been neutralised. We then wait for the collagen to dry before washing the layer with PBS to neutralise the acidity. Keratinocytes are then seeded on top of the collagen layer. As mentioned before, we waited again for the cells to adhere to the layers before adding DMEM to prevent the keratinocytes from being exposed to air. However, it was found that our cells have dried up before we are able to cover them up with the DMEM  likely due to the low volume of cell suspension we have added as mentioned by another member of the lab. He also took a look at the keratinocytes that we have been culturing and suggested that they may be differentiating due to their high confluency and suggested that we passage them at lower confluencies in order to prevent the keratinocytes from differentiating which will cause them to cease proliferating. Therefore, we have decided that we should culture more flasks of the keratinocytes in order to ensure we have sufficient number of cells for the experiments instead of passaging them at high confluencies to obtain more cells. We also decided to again redo our gelatin and collagen experiments .

Results: We kept the wells that we have prepared in the incubator even though they were not prepared correctly as mentioned in the paragraph above. As expected,the results were not very conclusive with little change in shape in the cells.  Moreover, Prof Tan also suggested that we should be using 0.02N of acetic acid instead of 0.5M of acetic acid to dissolve the collagen.

Second Gel Layer Experiment

The gelatin wells were again, prepared in the same way. However, for the seeding of the keratinocytes, we used a higher cell suspension volume in order to prevent the cells from drying up. For the collagen, we dissolved it in 0.02N of acetic acid instead of 0.5M of acetic acid. Prof Tan also suggested that for the assembly of the sandwich itself, the keratinocytes should be suspended in the collagen instead of on the collagen layer as it can be more easily transferred onto the gelatin-fibroblast layer. However, for this method, the collagen would have to be neutralised via the NaOH method instead of by PBS since the cells are no longer just in contact with the top surface of the collagen layer. Therefore we prepared the collagen wells according to this method. However, due to an oversight,  we did not manage to repeat the experiment for testing the adhesion of keratinocytes to collagen which involves the method of neutralising the collagen with PBS instead of NaOH which we will be doing in the following week.

Results: From the results we concluded that keratinocytes do not adhere to gelatin as there are no change in shape for keratinocytes in the gelatin well. The keratinocytes were found to have change their shape when suspended in the collagen layers that are soaked in DMEM, however, not enough of them are adhered to the collagen and hence they are not yet ready for the stratification stage. Therefore, there are two things suggested to us by Prof Tan and the other people at the lab. We will either have to decrease the seeding density for the keratinocytes so that they are not overcrowding or we will have to increase the collagen concentration to allow more keratinocytes to adhere.

Assembly of small peristaltic pump to machine and other modifications

The small peristaltic pump was mounted onto the machine via brackets that were 3D printed in the MnT lab. We also tested the ability of the machine to deposit gelatin and alginate onto a rectangular mould. From these tests, we identified several problems. We realised that our machine is not very flexible in terms of the printing process. The position of the mould cannot be moved, otherwise it will throw off the printing process. We also found out that trying to get an even layer on the mould is extremely difficult due to the high visocity of the alginate and gelatin. Therefore, we increase the number of lines for the printing process in order to minimise the dependence on the flow of the gelatin and alginate to achieve an even layer. We also 3D printed a mould as we find that the mould has a small protrusion that interferes with the formation of an even layer and a holder for the mould to ensure that the mould position will remain the same. A new nozzle holder was also designed and printed to integrate a scrapper that will help to even out the gel layer. In the following days we will test out the machine with these newly printed parts and further optimise the printing process.

 

 

 

Progress of Project for Week 10:

Gel Layer Experiment

As mentioned previously, we decided to try increasing the concentration of collagen used and also try out different seeding densities for the keratinocytes. In our previous experiments, we used a seeding density of 8*10^5 cells/well which results in the overcrowding and an earlier experiment using 2*10^5 cells/well results in there being too little cells to reach confluency. Therefore, we have decided to seed at 3 different densities, 3*10^5 cells/well, 5*10^5 cells/well and 7*10^5 cells/well. For the concentration of collagen, we decided to increase the concentration from 50ug/ml, using 50ug/ml, 100ug/ml, 200ug/ml and 400ug/ml collagen for the experiments. In total we used up all 12 wells of a 12 well plate.

Results: The keratinocytes changed shape and reached confluency near the periphery of the wells for all the concentrations of collagen. However, it was found that there are increasing number of dead cells as the concentration of collagen increases, especially at 200ug/ml and 400ug/ml. No significant difference was found between wells with different seeding densities. According to Prof Tan, this experiment is a success and hence what we should be doing next is to assemble the full sandwich with the keratinocytes and fibroblasts layers together, keeping our collagen concentrations between 50ug/ml and 200ug/ml and our seeding densities between 3*10^5 cells/well and 7*10^5 cells /well.

Gel Layer Experiment 2

For the full sandwich, we designed a total of 4 wells. From all our previous experiments, we learnt that 30% gelatin that we used for our experiments will melt in the 37 degree celcius incubator. The keratinocyte collagen layer is also relatively liquid due to the low concentration of 50ug/ml that we used for our experiments. This will then likely result in the keratinocyte collagen layer to sink through the liquidy gelatin layer and mix with the fibroblast gelatin layer. It is also likely that fibroblasts adhered to the gelatin might end up on top of the keratinocyte collagen layer. Therefore, to try to prevent this, we come up with the idea to add an additional alginate layer in between the gelatin fibroblast layer and the keratinocyte collagen layer. For the first well, we did not add the additional alginate layer and we did not add any fibroblasts to the gelatin layer. This is to test if the keratinocytes in the collagen could still change shape without a solid alginate layer below the keratinocytes. Fibroblasts were not added as they are similar in morphology to keratinocytes under a light microscope. For the second well, we added an additional alginate layer but we still did not add any fibroblasts to the gelatin layer. This is to test if the additional alginate layer could indeed prevent the keratinocytes from sinking to the bottom of the well and mixing with the fibroblast gelatin layer. For the third well, we decided to add both the alginate layer and fibroblasts to test if the sandwich can work with the additional alginate layer while for the fourth well, we added the fibroblasts but not the alginate layer to test if the sandwich really require the additional alginate layer to work. However, we do not have enough keratinocytes for all four wells and hence only performed the first 3 wells mentioned.

Results: None of the wells show a change in shape in the keratinocytes or fibroblasts. We theorised that this is likely due to the mixing of the gelatin and collagen layers and that the alginate layer by itself is insufficient to prevent this mixing of the gelatin and collagen layers.

Gel Layer Experiment 3

After consulting Prof Tan, we came up with several experiments. For the first experiment, we have an alginate bottom layer followed by a gelatin-collagen fibroblasts layer. This is because even if the gelatin melts it is likely that it would sink down and cover the alginate layer while bringing along the fibroblasts that have adhered to it, leaving behind a collagen layer that keratinocytes can still bind to. In the event that this gelatin-collagen layer does not melt, it also solves our problem of this layer mixing with the keratinocyte collagen layer. For the second experiment, we have a bottom layer made out of alginate,collagen and gelatin mixed with fibroblasts. This is to test if a layer made out of alginate, collagen and gelatin can remain solid to hold the fibroblasts while preventing the keratinocyte collagen layer from mixing with this layer. For our third experiment, we made a well using our original sandwich model, with alginate at the bottom, followed by gelatin fibroblast layer and then keratinocyte collagen layer. However, this time we use 40% gelatin instead of 30% gelatin and incubate the sandwich model at 32 degree celcius instead of 37 degree celcius as we have found out previously that 40% gelatin seems to remain solid at 32 degree celcius incubation. For our fourth experiment, we use a mix of collagen and alginate as our bottom layer and then add the keratinocyte collagen layer to the top of this layer. This is to test if the keratinocytes do sink to the bottom of the well, whether or not they are able to bind to a layer made out of alginate and collagen. If this is successful, then it might be possible to keep the fibroblast layer as gelatin only. The difference between this and the first experiment is whether or not collagen should be added to the bottom alginate layer or the fibroblast gelatin layer for the keratinocytes to adhere. For our fifth experiment, the bottom layer is made out of a mix of alginate, collagen and gelatin and the next layer is the keratinocyte collagen layer. This is to test if, in the event that the keratinocytes sink to the bottom, whether or not it is able to bind to a layer made out of alginate, gelatin and collagen. This will give us an idea of whether or not the second experiment would work without having to wait another few days after adding on the keratinocyte collagen layer. For the last experiment, a keratinocyte-collagen layer is incubated at 32 degree celcius to investigate if keratinocytes can still survive and bind to the collagen at 32 degree celcius.

Results: For the first experiment, it was found that the fibroblasts adhered well to the gelatin-collagen just as we expected and the gelatin melted. For the second experiment, it was also found that the fibroblasts adhered to the alginate-gelatin-collagen and the layer remain relatively solid to hold the fibroblasts. For our third experiment,  the fibroblasts still adhered to the gelatin but it was found that the gelatin layer has melted at 32 degree celcius after a few days. However, upon removal of the melted gelatin, it was found that there is still a thin layer of gelatin with the fibroblasts still adhered. For the fourth experiment it was found that the keratinocytes do not adhere when the bottom layer is alginate-collagen and they clump up together to form big lumps of cells. For the fifth experiment, it was found that the keratinocytes do not adhere when the bottom layer is alginate-collagen-gelatin, however, they remain healthy and are surviving well unlike in the fourth experiment where the cells are clumping up and appear to be dying. For the last experiment, it was found that the keratinocytes can adhere and survive very well within the collagen layer at 32 degree celcius. From these results, we conclude that we should try to put together our original sandwich model again with the alginate layer at the bottom, followed by the gelatin fibroblasts layer and then the keratinocyte collagen layer. However, this time round, we will be using 40% gelatin instead of 30% gelatin and incubating it at a lower temperature of 32 degree celcius instead of 37 degree celcius.

Calibration of large and small pump at LKC

Even though we have yet to sterilise the machine and bring it into the biosafety cabinet to try out the printing process. We have successfully calibrated both the big pumps and the small pumps to deposit the gel layers, calcium chloride and keratinocyte collagen solutions. Now all that is left to do is to test out the entire printing process within the biosafety cabinet. Even though at this stage, we are still not successful with regards to combining both our keratinocyte collagen layer and fibroblasts gelatin layers in a way that they can progress towards skin formation, we have shown that it is possible for both of them to grow and adhere to collagen and gelatin respectively. In the following week, we hope to be able to obtain some results for our combination of both of these layers. However, even if this is unsuccessful, it is only a matter of time before we find the solution to this problem. What is most important is that we have proven that it is indeed possible to “print” a bilayered living skin construct with a machine at far higher efficiency and lower costs compared to current methods.

Progress of Project for Week 11:

Gel Layer Experiment

As mentioned earlier, we made our original sandwich using 40% gelatin and incubate it at 32 degree celcius in a 35mm well. We also made 2 replicates with alginate-collagen-gelatin mixed with fibroblasts as the bottom layer and collagen-keratinocyte as the top layer as we found from our previous experiments that they remain relatively solid even at 37 degree celcius and the fibroblasts adhere well within the alginate-collagen-gelatin layer. Even though the keratinocytes do not seem to adhere well when the bottom layer is alginate-collagen-gelatin, they survived well and may adhere with longer duration of incubation.

Results: For the original sandwich, it was found that a small number of fibroblasts adhere to the gelatin layer. However, for the keratinocytes, it seem that most of them had lysed. This was also observed in the wells with alginate-collagen-gelatin as the bottom layers. We eventually concluded that these results might be due to some problems with our cells as we found out that our stock fibroblasts have all been contaminated and the incubator that we have been using seems to have a contamination problem. Hence, we are unable to obtain any results from this set of experiments.

Sterilizing of Cell Layering Machine

All equipment is sprayed down with 70% ethanol before being placed into the bio-safety cabinet for exposure to UV. For the tubings used for the extrusion of the alginate layer and the gelatin-fibroblast layer and the depositing of the liquid keratinocyte-collagen layer, 70% ethanol is passed into the tubing before washing away with PBS (phosphate buffer saline). They are then exposed to UV in the same cabinet. For the sterilization of the water bath, the water inside the water bath is emptied before the inside is sprayed with 70% ethanol. MilliQ water that are exposed to UV as well for sterilization is then used for the water bath.

 

 

Conclusion:

Even though we are unable to obtain any results for our last set of experiments, we are certain that we are very close to obtaining the desired results. We have already proven that the NiH-3t3 fibroblasts are able to grow and adhere in food grade gelatin and the HACAT keratinocytes suspended in 50ug/ml collagen are able to adhere and reach confluency at both 32 and 37 degree celcius when they are grown separately. The only thing that remains unresolved is putting both layers together in such a way as to allow both fibroblasts and keratinocytes to remain adhered to their respective layers while continuing to grow. Once this is also accomplished, the keratinocytes can then be exposed to air to promote differentiation and the stratification process for the formation of the epidermal layer of the skin while the fibroblasts secreting the ECM (extracellular matrix) molecules will form the dermal layer.