This protocol offers an in vitro strategy to deliver stem cells from chitosan microspheres into a collagen gel scaffold, harvest and culture. The primary adipocyte stem cells then emulsify and recover a preparation of chitosan microspheres. Next, seed the stem cells onto the chitosan microspheres and culture them in an eight micron pore size membrane culture plate insert to produce a CS loaded CSM finally mix the microsphere delivery system with a three dimensional collagen gel scaffold.
Ultimately, this CSM system offers potential for use in regenerative medicine and drug delivery. The main advantage of this technique over existing methods like cell injection or microencapsulation, is the ability of cells to attach to a carrier material and persist after initial application. Generally, individuals new to this method will struggle because esan microspheres has an inherent static charge.
And loading cells over a microsphere bed will be challenging without disturbing them. Excise and wash, perineal and epidermal adipose tissue from a wrap mince the tissue, then transfer one to two grams into 25 milliliters of HBSS containing 1%FBS. After centrifugation, collect the free floating adipose tissue layer into a 125 milliliter erlenmeyer flask.
Add 25 milliliters of collagenase type two and incubate reaction on an orbital shaker. Now carefully remove the liquid fraction below the oil and adipose layer and filter sequentially through nylon mesh filters of 170 microns. Harvest the cells by centrifugation after two washes with 25 milliliters.
HBSS Resus, suspend the pellet in 30 milliliters of supplemented mesen pro growth medium. Split the purified adipose derived stem cells into two T 75 culture flasks and amplify the A SC preparation in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. This CSM protocol is a water in oil emulsification process that utilizes an ionic coer technique.
Emulsify six milliliters of an aqueous solution of chitosan in 100 milliliters of an oil phase mixture using overhead and magnetic stirring simultaneously in opposite directions for one hour to the stable water and oil emulsion, add 1.5 milliliters of 1%potassium hydroxide in n octal every 15 minutes for four hours. After completion of the ionic cross-linking reaction, slowly decant the oil phase and immediately add the spheres to 100 milliliters of acetone. Wash the spheres with acetone until the oil phase is completely removed.
Then dry the recovered spheres in a vacuum. Desiccate determine the average CSM particle size surface area per milligram and the unit cubic volume using particle size analyzer. Proceed to wash CSM three times with sterile water to remove residual salts.
Now sterilize the CSM preparation with absolute alcohol for use in cell experiments in a 50 milliliter glass tube, mix five milligrams of microspheres with one milliliter of 0.5%TNBS solution incubate for four hours at 40 degrees Celsius. Then add three milliliters of six normal hydrochloric acid and incubate at 60 degrees Celsius for two hours. After cooling the samples to room temperature, add five milliliters of deionized water and 10 milliliters of Ethel Ether.
To extract the free TNBS remove a five milliliter aliquot of the aqueous phase and warm to 40 degrees Celsius in a water bath for 15 minutes to evaporate any residual ether after cooling to ambient temperature. Dilute with 15 milliliters of water with a spectrophotometer. Measure the absorbance of the prepared chitosan microspheres at 345 nanometers using TNBS as blank.
Also measure the chitosan used for the CSM preparation. Now, calculate the number of free amino groups of the CSM relative to the total number of amino groups in chitosan. Equilibrate five milligrams of the sterilized CSM in sterile HBSS overnight.
Transfer the CSM to an eight micron membrane insert for 24 well culture plates. After the CSM have settled, carefully aspirate the HBSS. Now add 300 microliters of growth medium to the inside of the insert, and 700 microliters of growth media to the outside of the insert.Resus.
Suspend the adipose derived stem cells in 200 microliters of growth, medium and seed over the CSM inside the culture plate. Insert incubate for 24 hours in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. Transfer the a SC loaded CSM into a sterile micro centrifuge tube without disturbing the cells that have migrated into the insert membrane.
Now remove the residual medium and add 250 microliters of fresh growth medium to each tube. Add 25 microliters of five milligrams per milliliter MTT solution and incubate for four hours. Remove the media and add 250 microliters of dimethyl sulf oxide vortex to solubilize the form asan complex centrifuge, the CSM at 2, 700 G for five minutes.
Measure the supernatant absorbance at 570 nanometers using 630 nanometers as reference. Proceed to determine the cell number associated with the CSM relative to the absorbence value obtained from known numbers of viable A SC.Mix five milligrams of a SC loaded CSM with 7.5 milligrams per milliliter. Type one collagen extracted from rat's.
Tail tendon, adjust the pH to 6.8 using two normal sodium hydroxide and pour into a culture plate. Insert to fibrillate now transfer to a 12 well plate and incubate for 30 minutes. Continue to refresh the medium for the collagen A-S-C-C-S-M gels every other day of the incubation period.
Also monitor, release and migration of cells from CSM into the gel using standard microscopy techniques. This following example describes the in vitro strategy to deliver adipose derived stem cells from chitosan microspheres into a collagen gel scaffold. SEM and TEM images indicate a preparation of porous CSM of uniform size.
Incubation of the microspheres with primary adipose derived stem cells indicates 4, 000 cells per milligram of CSM calcium am. Staining of a SC shows spreading over the CSM with extending filopodia into the porous crevices of the microsphere. When the cell loaded CSM were mixed with the collagen gel, the cells migrate into the gel and can be followed over time.
The implications of this technique immediately extend towards the therapy of massive and chronic tissue loss. However, this treatment regimen has much more potential due to its versatility to integrate with a variety of cells and or biomaterials used to treat different levels of soft and heart tissue losses. Doubt this method can provide insight into host cell delivery.
It can also be applied to other systems such as injectables, delivering therapeutic drugs, developing hybrid systems to deliver both cells and therapeutic drugs together.
