The overall goal of the following experiment is to create an ex vivo model that replicates normal and abnormal hematopoesis. This is achieved by fabricating a scaffold made of polyurethane using the thermally induced phase separation technique or tips, which produces a highly porous, non biodegradable scaffold in the form of a Petri dish. Next, the scaffold is cut into cubes and is then coated with extracellular matrix proteins to aid cellular adherence When seeding the cells, the scaffolds are sterilized using UV and ethanol, and they are incubated in media until seeded.
The next step is to isolate mononuclear cells from cord blood samples, which are used to seed the scaffolds. Results have shown that this model supports the growth of human cord blood mononuclear cells and primary leukemic cells in the absence of exogenous cytokines based on data from M-T-S-S-E-M and flow cytometry assays. This method can help to answer key issues in dermatology field, such as creating an experimental model of the bone marrow to study cell interactions, and also to explore novel treatments.
In leukemia, Though this method can provide an ex vivo biomimicry of the bone marrow. It can also be applied to other systems such as neuronal cells for the in vitro reconstruction of the neuron network as a model to understand the natural neuronal outgrowing and also to study neurological disease. The implications of this technique extend towards therapy of blood-related disorders such as leukemia because different drugs can be tested towards their treatment in an environment similar to the human body.
To fabricate PU scaffolds in the form of 90 millimeter Petri dish discs, use the thermally induced phase separation process. Briefly prepare 60 millimeters of polymer solution per scaffold. Pour the solution into the dish molds 0.5 centimeters deep, freeze the molds and allow for solvent sublimation.
Then set up the tips equipment after freezing, cut the scaffold discs into 0.5 centimeter cubes. Prewitt the scaffold cubes by immersing them in 70%ethanol for one minute, then transfer them to PBS for 20 minutes. After the PBS bath load 20 cubes into 50 milliliter conical tubes and centrifuge the scaffolds.
Next, add 40 milliliters of ECM protein solution to each conical and centrifuge them.Again. Now remove the protein solution and add 40 milliliters of PBS to each conical and centrifuge them once more. Now unload the scaffold cubes to 24 Well tissue culture plates placing one scaffold in each well to sterilize them, subject them to an eight minutes of UV light, followed by immersion in 70%ethanol for two hours after that.
Wash the scaffolds twice in PBS and then submerge them in supplemented IMDM. Store them in an incubator until they are seeded. When the scaffolds are ready, extract human MNCs from samples of cord blood or leukemic bone marrow.
Begin with a fal paque density gradient. Centrifugation of the tissue in 50 milliliter conical tubes. Collect MNCs from the suspension.
Then count the cells and resus, suspend them in. Im DM with the MNCs prepared seed. 2 million cells in 100 microliters of solution onto each scaffold.
Incubate the seeded scaffolds for 10 minutes so that the cells can settle into the scaffolds. After 10 minutes, fill each well with 1.5 additional milliliters of supplemented IMDM and return the plate to the incubator. For the duration of the experiment on a daily basis, replace all the medium.
The MTS assay measures cellular metabolic activity in relation to absorbence. Place one unseed scaffold and two seated scaffolds. In a 24 well plate add one milliliter of media and 200 microliters of the MTS solution to each.
Well incubate the plate for three hours at 37 degrees Celsius. Next, transfer 100 microliter samples from the supernatant into eight wells of a 96 Well plate. Complete the assay by measuring absorbance at 490 nanometers.
Using a microplate reader at different culture time points, remove one scaffold seated with MNCs from the media. For SEM analysis, fix the scaffold with 2.5%PBS buffered glu aldehyde solution for 40 minutes at four degrees Celsius. Then wash the fixative off twice with PBS dehydrate, the scaffold in a graded ethanol series, allowing 10 minutes for each submersion.
Remove the sample from 100%ethanol and allow it to dry at room temperature for four hours. Now section the specimen manually using a scaffold, making sure that the scaffold is cut with the pores facing up. Mount the sections on glass on metal holders for SEM.
Then sputter coat the slides with gold to prepare for SEM evaluation for cyto spins at different time points. Remove the scaffold from culture and aspirate cell samples for cytology. Make sure that you aspirate as much media as possible from the pores of the scaffold.
After using a standard cyto spin mounting technique, stain the slides with modified right gim. Sustain for six seconds, followed by a wash in distilled water for 10 minutes and observe them under a microscope at different time points. During the incubation.
Remove a seated scaffold and fix it with ethanol vapor overnight. The next day, air dry the fixed scaffold and freeze it until all the required scaffold time points are collected. For analysis section each scaffold into 30 micron fixed slices and slide mount them for processing.
Block the samples in PBS with 10%fetal bovine serum for 30 minutes at room temperature. Then submerge the samples in a one to 50 dilution of primary monoclonal antibody and incubate them overnight at four degrees Celsius in darkness the next day. Wash the samples three times in PBS and stain them with a one to 200 dilution of secondary antibody for one hour.
At room temperature in darkness, wash the antibodies off the samples three times with PBS when the baths are completed. Observe the slides under a confocal microscope using a watery emission 60 x objective lens and an excitation frequency of 488 nanometers for flow cytometry. Aspirate 250 microliters of cell suspension from a seated scaffold at different time points and spin them down.
Aspirating the cells out from the scaffold. It's not very easy because the cells have penetrated inside the scaffold through the interconnected pores. So to extract the cells as viable as possible, make sure that you don't treat the cells too harsh while trying to get as much media as possible from inside.
The scaffold. Resuspend the cell pellet in 80 microliters FC buffer, which should provide a density of about 1 million cells per milliliter. Add 10 microliters of each antibody fluorescent die to the cell suspension for MNC detection.
A combination of three antibodies is used now incubate the cells for 30 minutes at four degrees Celsius in the dark. After centrifusion, wash the chilled cells twice with one milliliter of PBS and finally, resus suspend them in one milliliter of FC buffer. The cells are now ready for flow cytometry.
Due to the heterogeneous nature of hematopoiesis. Two different cell types were used. Hematopoietic cellular growth, kinetics of normal and abnormal hematopoietic cells.
Grown without growth factors was measured by the MTS assay after just seven days in culture. Cellular proliferation of human C-B-M-N-C cells was evident. Human primary leukemic cells, however, grew more slowly.
Nevertheless, both cell types grew and provided a model. After 28 days of culture, the morphology of the hematopoietic cells and leukemic cells was observed by cytology and SEM. Central sections of the scaffolds were analyzed and showed the spreading of the seeded cells throughout the scaffold, establishing themselves in clusters and in niche like structures.
The pore size and distribution in an unseeded scaffold was used as a control after 28 days. Multi photon microscopy was used to highlight the distribution of the cells within the 3D scaffold in situ. This revealed the presence of erythroid islands in central sections of the scaffold by the expression of the markers CD 71, which is positive in erythroblasts.
This proves the importance of mature and maturing cells during erythropoiesis. Finally, flow cytometry graphs of the cells prior to seeding shows a difference in the phenotype of hematopoietic cells. Levels of CD 2 35, A positive and CD 45 positive corresponding to erythrocytes and leukocytes respectively are higher in the normal sample than in the leukemic sample.
Highlighting the hemo blast nature of the leukemias. While attempting this procedure, it is important to remember to distinguish the different properties of the cell Sitting. Extracting the cells is a crucial part of the protocol, and some cells may attach to the scaffolds more than others.
Try to maintain cell viability high by aspirating the cells gently. After watching this video, you should have a good understanding of how to produce an exvivo model of normal and abnormal hematopoiesis. This includes the formation of treatise, scaffolds followed by sterilization, seeding, culture harvesting, and finally, analysis of the cells Following this procedure.
Other metals like western bloating of proteins produced by the cell in culture or polymerase gene reaction of the cellular DNA can be performed in order to answer additional questions like, which type of soluble or cell associated factors or genes are involved in the ex vivo cell growth and differentiation.
