The use of injectable spherically symmetrical cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation
Wen-Yu Lee, Yu-Hsiang Chang, Yi-Chun Yeh, Chun-Hung Chen, Kurt M. Lin, Chieh-Cheng Huang, Yen Chang, Hsing-Wen Sung
Summary:
Generally, cell transplantation at sites of muscular tissue injury involves directly injecting dissociated mesenchymal stem cells (MSCs) into the tissue. While some cells are able to attach and begin repairing the cell graft sites, many do not localize specifically at the injury site and optimal muscle repair cannot be achieved. The authors of this paper designed an approach in which they first created spherical aggregates of MSCs in vitro and then injected it into the damaged skeletal muscle of mice to see if it made a difference in the efficiency of transplantation. To judge the efficacy of their cell aggregate transplantation delivery system, they compared cell engraftment of the aggregate to dissociated cells as a control.
Before beginning the cell transplantation experiments, the authors first created different sized MSC aggregates by seeding different cell densities (5.0E3, 1.0E4, 5.0E4, 1.0E5 and 2.0E5 cells/well) in 96-well methylcellulose (MC) hydrogel systems with a multichannel pipette. The bone-marrow MSCs were extracted from the femora and tibia of FVB green-fluorescent transgenic mice that could express enhanced green-fluorescent protein (EGFP) to make visualization easier. The single cell aggregates that formed via cellular self-assembly in each well were imaged to determine the diameter of the spherical aggregates prior to injection. Cell aggregates that were seeded at 1.0E4 cells/well density had a 195 +/- 15 um diameter. They tested the viability of the cell aggregates using a live/dead assay; cells that died would no longer have membrane integrity and would stain red if not viable. Based on their fluorescence images of optical sections (Fig. 5 on right) most of the cells were viable. When seeded at 5.0E4 cells/well or greater, the radii were greater than 200 um and the cells that were located in the center were difficult to image due to the penetration limit of the confocal laser light (~100 um from surface). These cell aggregates were considered not viable due to the fact that the cells in the center would most likely develop hypoxia since they are beyond the diffusional capacity distance of oxygen, which is around 200 um. Additionally, cells seeded at 5.0E4 cells/well density did not remain intact after being injected through a 27-gauge needle, whereas the 1.0E4 cells/well seeding density aggregates did. Therefore, they chose to inject 1.0E4 cells/well seeding density cell aggregates for their in vitro and animal studies.
To test whether the injected cell aggregates retained activity after injection through a needle, they plated the cell aggregates after transferring them from their original culture medium via a 27-gauge needle. They discovered that the cell aggregates adhered to the plates shortly after seeding and cells migrated out of the cell aggregate, attached to the culture plate, and proliferated. With this, the authors decided to test the cell aggregates in vivo and transplanted them into the left thighs of transgenic mice. They stained for BrdU to label the cells of interest and they noticed a greater amount of incorporation of transplanted cells in the damaged host tissue as compared to the control disperse cell transplantation. This increase in attachment and proliferation is mainly due to the inherent extra-cellular matrix (ECM) that provided a better environment for the MSCs to transplant successfully. When compared to the dissociated cells that were injected in the right thigh as a control, there were only a few BrdU-labeled cells, which suggests that a majority of the transplanted individual cells were washed out after the injection due to their small size.
Figure 8: Immunofluorescence images of test samples obtained from the groups treated with dissociated cells or cell aggregates retrieved at day 1 or 4 weeks postoperatively.
Conclusion and Final Thoughts:
The authors presented a direct approach to create spherically symmetric cell aggregates that maintained their endogenous ECM upon injection into damaged muscle. They demonstrated through their in vivo animal study that the cell aggregates did populate the graft site more effectively than the typical dissociated cell transplantation. This paper reports a great improvement on the typical cell transplantation technique into infarcted muscles, but is not without faults. They reported an inflammatory response that occurred within the first day of the intramuscular injection with a nearly 20% increase in macrophage presence at the site of injection. Even after 4 weeks postoperatively, there was still a decent amount (~5.4%) of macrophages at the site, which decreases the efficacy of the transplanted cells since they are getting attacked by the host. To improve this, they could try implanting MSCs derived from the host animal itself to reduce the immune response and possibly improve the cell graft incorporation.
7 comments:
These cell aggregates were considered not viable due to the fact that the cells in the center would most likely develop hypoxia since they are beyond the diffusional capacity distance of oxygen, which is around 200 um.
Do you know how they come up with this number? Do they do any math, or is it a ballpark amount?
So were the cells within the hydrogel and the entire hydrogel-cell system implanted into the subject? Also, how did they thermally-activate the hydrogels during the in vivo animal testing? I’m assuming that they heated the region with some sort of external instrument that warmed the treated area, expanding the hydrogel to fill the damaged muscle, but this is just my guess.
Another question I have is if the authors mentioned how long it took for the hydrogel to degrade within the body and how degradation affected the efficiency for the cells to attach to the tissue? If the gel degraded too fast, the cells may not have enough time to adhere to the muscle surface that needs to be repaired.
Terry: The authors approximated the diffusional capacity and cited it from another paper without doing any calculations or tests on their own.
Brian: The hydrogel was not actually implanted in the animals, it was used to create spherical cell aggregates prior to implantation. The cell aggregate itself was later implanted in the subject. As the hydrogel was not injected into the subjects, they did not mention the degradation rate of the methylcellulose hydrogel in vivo.
Why were the other cell densities (5e3, 1e5, 2e5 cells/well)not used to create the cell aggregates for injection? Also could lower cell densities decrease the inflammatory response but still be sufficient for transplantation?
They wanted to use the largest cell aggregate that was still viable in order to maximize the number of cells that could localize to the site of injury. As a lower cell density would most likely decrease the inflammatory response, incorporation of transplanted cells would not be as efficient since fewer cells would be able to attach.
I feel like the spherical symmetry part did not help the transplantation at all, it could have been any arbitrary shape, as long as the cell were in some form of aggregate with some extracellular matrix holding them together. for example, a multilayered cell surface could have the same effect and it will not run into the hypothia problem of oxygen.
I understand that the reason that the researchers did not use other cell densities (5e3, 1e5, 2e5 cells/well was because of efficiency. However, for future work, i feel that the researchers should inject smaller cell densities repeatedly (with some time delay) and compare the inflammatory response with a large injection of cells.
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