2015 Fiscal Year Annual Research Report
移植医療を標的とした細胞組織を封入するためのマイクロ流体システムの開発
| Project/Area Number |
14F04771
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| Research Institution | The University of Tokyo |
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Principal Investigator |
竹内 昌治 東京大学, 生産技術研究所, 教授 (90343110)
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| Co-Investigator(Kenkyū-buntansha) |
MAZARI-ARRIGHI ELSA 東京大学, 生産技術研究所, 外国人特別研究員
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| Project Period (FY) |
2014-04-25 – 2017-03-31
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| Keywords | セルファイバ / Microfluidics / 肝細胞 / 三次元組織構築 / 細胞外マトリクス / 再生・移植医療 |
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| Outline of Annual Research Achievements |
We found that a core composed of primary liver cells at 9.107 cells/mL in a mixture of native collagen supplemented with Matrigel enable to generate hepatic microfibers that are self-assembled on day 2. We analyzed hepatic cells viability and preservation of liver functions compared to 2D conventional culture over 7 days. Measurements on 3D microfibers compared to the ones on 2D conventional culture indicated a higher density of viable cells (> 65%), an enhanced albumin production (around 6 times higher), a high CYP1A1 secretion in 3D (10 fold higher than in 2D), as well as increased urea synthesis (around 8 times higher). It is worth to note that hepatic cell microfibers maintained high liver-specific functions activities of cultured hepatocytes until 3 weeks showing that these microconstructs are a pertinent microenvironment for primary hepatocytes culture.
Furthermore, regarding proliferation assays, we seeded hepatic cells at 2.5.107 cells/mL in native collagen with Matrigel. After 2 days of culture, we supplemented culture medium with 50% of 3T3 cell microfiber conditioned medium. We monitored cell viability, albumin secretions, and urea synthesis during 3 weeks. Such measurements performed on induced hepatic microfibers compared to control hepatic ones highlighted that after 1 week of culture the total cell number is 2.5 times higher, albumin secretions are 10 times higher, and also urea synthesis is increased by 2. Such induced hepatic cell microfibers were able to be maintained in culture with significant liver-specific secretions during at least a 3 weeks.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We developed an isolation protocol that enabled us to efficiently collect rat primary hepatocytes with a high cell viability. Using cell fiber technology, we were able to encapsulate and to culture in vitro rat primary hepatic cells into core shell microfibers. By performing various in vitro assays, we also characterized the properties of hepatic cell microfibers such viability and liver-specific functions. Finally, by optimizing different culture condition parameters, we were able to maintain our hepatic cell fibers during a long-term period.
Furthermore, we've set up and started in vivo assay in hepatitis model rat by using the established system. We are progressing to evaluate the function of the hepatic microfibers.
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| Strategy for Future Research Activity |
First, during this project, we always used very high-viability and high-purity suspension of primary hepatocytes. Actually, it means that the average cell viability is over 90% and within these suspensions fluorescent-activated cell sorting experiments indicate over 90% of cells are hepatocytes. However, such experiments did not mimic in vivo-like liver cell population that is basically around 80% of hepatocytes and 20% of non-parenchymal cells. That is why, we plan to fabricate cell hepatic microfibers using non-purified and then to record properties of these new types of microfibers such as cell viability, cell types, albumin, urea, and CYP1A1.
Then, instead using rat primary hepatocytes, we will fabricate thanks to our encapsulation technique human primary cell microfibers and we will try to make liver human cells proliferate using our dedicated hepatic cell culture protocol.
Finally, we already characterized cell hepatic microfibers properties in vitro but as a proof of concept we plan is to demonstrate the relevance of such microstructures in vivo. In order to do so, we will continue to transplant hepatic cell microfibers into Nagase analbuminemic rats (NARs). These rats have a very low blood albumin level (under 1mg/dL while for normal rat 3.3g/dL) and we expect to monitor an increased of blood albumin in transplanted rats, suggesting that our hepatic cells microfibers have potential for use in the clinic.
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