Different mechanical loading parameters could induce tendon-derived STEM cells differentiated into different cells. We introduce detailed process of applying this three-dimensional uniaxial mechanical stimulation bioreactor system to induce tenogenic differentiation of tendon-derived STEM cells. Tendinopathy is one of the common sports injuries in the orthopedic department.
The presence of Achilles tendinopathy is most common with up to 29%for middle and long-distance runners, while the presence of patella tendinopathy is also high in athletes of volleyball, basketball, track and field, handball, and soccer. However, due to the limited self healing ability of tendon and the lack of effective treatment, tendinopathy still has significant adverse effect on the life quality of patients. Tendon derived STEM cells, known as TDSCs as the important precursor cells of tendons cells, play an important role in the development of tendinopathy, as well as their functional and structural restoration after tendinopathy.
However different to mechanical loading parameters, such as loading stress, loading frequency, loading type, and loading period can induce TDSCs differentiate into different cells. Thus, an effective and valid mechanical loading regime is very significant for tendon genesis. There are seven stages of the present protocol, including isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon STEM cell construct, assembly of the uniaxial cells stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue.
Isolation of mice TDSCs. Used in mice that are six to eight week old mice by cervical dislocation. Harvest patella tendons and Achilles tendons.
Digest tendon with six milliliter Type 1 collagenase for three hours. Collect the cells and culture it in complete alpha MEM containing 10%of FBS and 1%of streptomycin and penicillin mixture for seven days. identify TDSCs using fluorescence activated cell sorting by flow cytometry, expression of cell surface markers, including CD44, CD90, and Sca-1.
Lack off the expression of CD34 and CD45. Passage and freeze cells. Passaged cells will be used for further steps.
Culture and expansion of mice TDSCs. Take the warmed up mice tendon STEM cells. Slowly add extra four to five milliliter of complete alpha MEM.
Transfer the mixture to a 15 milliliter centrifuge tube by a pipette. Top up with medium to a total eight milliliter by a pipette. Place the tube into centrifuge a well balance.
Centrifuge cells at 347 g for five minutes. Take out a tube from the centrifuge. Check the pellet at the bottom.
Decant the free medium. Re-suspend cells gently in one to two milliliter complete medium. Avoid making too many bubbles.
Transfer re-suspended cells to a T75 flask by a pipette. Use a pipette to add complete alpha MEM medium to the flask to reach a total volume of 10 milliliter with the final concentration of 13, 000 cells per square centimeter. Place the flask into the incubator and culture it at 37 degrees with 5%carbon dioxide.
Change the medium every three days. Observe and monitor the cells under a microscope when changing the medium until cells were cultured to 100%confluence. Preparation of stimulation culture medium for cell sheet formation.
Pour 15 milliliters complete alpha MEM medium into a 15 milliliter sterile tube. Add six microliter ascorbic acid into 15 milliliter medium to the final concentration of 4.4 microgram per milliliter. Mix gently by inverting up and down.
Cell sheet formation by culturing in stimulation medium. Discard non-normal complete alpha MEM medium carefully. Avoid touching the cells attached to the bottom of the flask.
Add 10 milliliters stimulation medium slowly. Avoid causing any disturbance to the fully confluent cells. Culture the cells in stimulation medium for nine days and change the medium every three days to sufficiently generate cell sheet at 37 degrees with 5%of carbon dioxide.
Preparation of 3D tendon STEM cell construct. Take the flask out of the incubator. Discard of the stimulation medium completely.
Wash the monolayer cell sheet with PBS by swirling the flask. Discard of the PBS completely. Use a pipette to add one milliliter, 0.25%trypsin to the corner of the flask.
Tap the corner of the flask to detach the monolayer cell sheet until the corner of the cell sheet washes off the bottom of the flask. Immediately add nine milliliter complete alpha MEM medium to stop the reaction of trypsinization. Continue swirling the flask to completely peel off the monolayer cell sheet.
Pour out the total to attach the cell sheet into Petri dish with medium. Use a sterile tweezer to pick up one corner of the cell sheet and rotate in a clockwise direction for 15 times. Pick up another end of the cell sheet and rotate in an anticlockwise direction for 10 times to formulate a tendon-like in vitro construct.
Assembly of the uniaxial-stretching mechanical stimulation complex in unique designed bioreactor. Connect the hooks by the connector, then adjust to two centimeters between two hooks. Gently wind the 3D TDSCs construct on the demo hook for three times on each hook.
Secure the hooks with cell construct onto the chamber of the bioreactor by tightening the screws on two ends. Fill up the chamber with the complete alpha MEM medium. Connect the accelerator to the culture chamber by cable.
Cut the hooks'connector by sterile tweezers. Switch on the power and channel controller to start a mechanical stimulation. Put the lid on the chamber, track the indicator lights, and assure the bioreactor can function properly.
Put the bioreactor in the incubator and subject the 3D cell construct to mechanical stretching. The loading regime is 6%strain, 0.25 Hertz frequency, eight hours followed by 16 hours rest for six days. Evaluation of the mechanical stimulated in vitro tendon-like tissue.
loosen the screws by a screwdriver and take assembly off carefully. Put the tendon-like tissue into 4%PFA for fixation for 15 minutes, Place the fixed tendon-like tissue into a biopsy cassette with biopsy from pets. Process to dehydrate a sample.
And finally, evaluate by HD histological standing. Repeat the whole protocol and extract RNA from the tendon-like tissue for evaluating the expression of tenogenic markers by qPCR. Before mechanical stimulation, TDSCs will grow to 100%confluence in complete medium and display a disorganized outward structural morphology.
After six days you next saw stretching, mechanical loading, extra cellular matrix, and the cell alignments were well orientated. Cells were well populated and well enveloped in ECM after mechanical loading. Cell morphology was presented to be located and was more similar to normal tendon cells compared to the one with without stretching.
Cell density in cell construct with loading was higher than the one without loading. qPCR results showed that the present matters increased the expression of tenogenic markers, including scleraxis, Mohawk, tenomodulin, and Type 1 collagen compared to the ones treated by stated culture. In terms of mechanical stimulation bioreactor system, separated chamber with linear motor was used in our present protocol.
Because it can provide accurate mechanical loading and it is portable and easy to be adjusted compared with the ways the traditional 2D cells sheet and the biaxial stretch, 3D cell construct is more similar to the real shape of tendon. And the uniaxial stretch is more similar to the strengths characteristics of tendon cells. This provides a theoretical basis of using 3D uniaxial stretched system to stimulate the differentiation of TDSCs.
Thus, 2D cell sheet were finally routed into a 3D cell construct and underwent uniaxial in the present protocol. According to our bioreactor system, our loading regime is 6%strain, 0.25 Hertz frequency, eight hours followed by 16 hour rest for six days. Finally, the effectiveness of the protocol was demonstrated by morphology and the expression level of tenogenic markers.
The whole operation process is simple, but the operator need to be pay attention to keep the environment of TDSC sterilized at all times. There are many applications of our protocol. The process of stimulating TDSCs to mimic differentiation in vivo is helpful for investigation of pathological process of tendinopathy.
Besides, this protocol provides a reproducible method for directed differentiation of TDSCs into tendon-like tissue. So it can be used simply and effectively for potential engineered tendon culture. In summary, applying a 3D uniaxial mechanical stimulation bioreactor system is simple, economic, reproducible, and a valid method to induce tenogenic differentiation of TDSCs.
Our protocol provides the whole process in details.
