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評估以不同製備方式之人類血小板釋放物培養擴增細胞應用於再生醫學
Other Title
Evaluation of various human platelet lysate fractions as supplements for ex vivo expansion of cells used in regenerative medicine applications
Type
thesis
Date Issued
2016-07-19
Author(s)
陳明昇
Advisor
白台瑞
Subjects
系所名稱：生醫材料暨組織工程研究所
Description
學位別：碩士
語文別：英文
指導教授：白台瑞
共同指導教授：
口試委員：王宗仁;陳盈汝
中文關鍵字：血小板裂解液;再生醫學;細胞治療;角膜內皮細胞;幹細胞
英文關鍵字：Platelet lysate;regenerative medicine;cell proliferation;BCEC;WJMSC
語文別：英文
指導教授：白台瑞
共同指導教授：
口試委員：王宗仁;陳盈汝
中文關鍵字：血小板裂解液;再生醫學;細胞治療;角膜內皮細胞;幹細胞
英文關鍵字：Platelet lysate;regenerative medicine;cell proliferation;BCEC;WJMSC
Abstract
Background
Clinical-grade cell ex vivo expansion has great potentials to replace transplant tissues in regenerative medicine. Ex vivo cells expansion procedures need a growth medium supplemented with nutrients, such as, fetal bovine serum (FBS). Using FBS is controversial due to safety concerns and unethical production methods. Pooled human platelet lysate (HPL), increasingly used as FBS substitute, has also drawbacks. HPL needs to be refined for optimal growth of specific cells and virally-inactivated to improve safety. Corneal endothelial cell (CEC) can increase the availability of corneal tissue for transplantation, but are hard to expand in vivo. Wharton's jelly mesenchymal stem cells (WJMSC) is an important source of stem cells for therapeutic applications. Optimal HPLs fraction supplements for these two types of cells should be developed and impact of viral inactivation on ex vivo cell expansion determined.
Aim
To study various HPL preparations as supplements of growth media for the ex vivo expansion of CEC and WJMSC.
Materials and methods
HPLs were prepared from platelet concentrates (N = 4) by three freeze and thaw cycles at -80/+37°C (FT), calcium chloride activation, subjected to heat-treatment at 56℃ (GBHPL) or not (GBPL), or viral inactivated by solvent/detergent (SD). HPLs were characterized by SDS-PAGE, Western blot, and growth factors measured by ELISA. BCEC and WJMSC were cultured in DMEM or α-MEM media supplemented with 10% or 20% of FBS (control) or supplemented with 10% or 20% of the different HPL materials at normalized protein content, and containing 3 unit/ml heparin (for SDPL and FTPL) to avoid fibrin formation. Cell morphology was observed by optical microscopy. BCECs cell viability was determined for up to 7 days using the MTT assay. Cell functional junction zonula occludens-1 (ZO-1) determined by Western blot. WJMSC doubling time was determined from passages 4 to 9; after 5 passages WLJMSC CD29, CD31, CD44, CD73, CD90 and CD105 immunophenotype markers were measured by flow cytometry (FCM), and potential of differentiation into the chondrocyte, osteocyte and lipocytes lineages evaluated.
Results
The HPL materials had different protein composition, Calcium chloride activation removed most of fibrinogen. The same trends in growth factor content by ELISA in the four HPL fractions was found. BCEC morphology revealed increased shrinking in medium supplemented with SDPL, whereas the other conditions were not different from the control group. BCEC viability was similar in all groups to that using 10% FBS. WJMSC cells became bigger (100 μm) in the control group after 5 passages whereas cells morphology remained unchanged in the media supplemented with each HPL fraction the same as 20% FBS protein content. After 5 passages in all HPL-supplemented media, WJMSC showed higher proliferation rate, maintained their immunophenotypic characteristics, and exhibited enhanced capacity to differentiate into the osteocyte, chondrocyte, and adipocyte lineages.
Conclusion
All HPL fractions, apart SDPL, could be used as supplements of growth media for expanding BCEC and WJMSC ex vivo based on cell viability and doubling time. In addition, BCEC cell junction markers were maintained. WJMSC immunophenotypes and differentiation capacity were preserved over several passages in HPL-supplemented media and showed healthier morphology than when expanded in FBS-supplemented medium.
Clinical-grade cell ex vivo expansion has great potentials to replace transplant tissues in regenerative medicine. Ex vivo cells expansion procedures need a growth medium supplemented with nutrients, such as, fetal bovine serum (FBS). Using FBS is controversial due to safety concerns and unethical production methods. Pooled human platelet lysate (HPL), increasingly used as FBS substitute, has also drawbacks. HPL needs to be refined for optimal growth of specific cells and virally-inactivated to improve safety. Corneal endothelial cell (CEC) can increase the availability of corneal tissue for transplantation, but are hard to expand in vivo. Wharton's jelly mesenchymal stem cells (WJMSC) is an important source of stem cells for therapeutic applications. Optimal HPLs fraction supplements for these two types of cells should be developed and impact of viral inactivation on ex vivo cell expansion determined.
Aim
To study various HPL preparations as supplements of growth media for the ex vivo expansion of CEC and WJMSC.
Materials and methods
HPLs were prepared from platelet concentrates (N = 4) by three freeze and thaw cycles at -80/+37°C (FT), calcium chloride activation, subjected to heat-treatment at 56℃ (GBHPL) or not (GBPL), or viral inactivated by solvent/detergent (SD). HPLs were characterized by SDS-PAGE, Western blot, and growth factors measured by ELISA. BCEC and WJMSC were cultured in DMEM or α-MEM media supplemented with 10% or 20% of FBS (control) or supplemented with 10% or 20% of the different HPL materials at normalized protein content, and containing 3 unit/ml heparin (for SDPL and FTPL) to avoid fibrin formation. Cell morphology was observed by optical microscopy. BCECs cell viability was determined for up to 7 days using the MTT assay. Cell functional junction zonula occludens-1 (ZO-1) determined by Western blot. WJMSC doubling time was determined from passages 4 to 9; after 5 passages WLJMSC CD29, CD31, CD44, CD73, CD90 and CD105 immunophenotype markers were measured by flow cytometry (FCM), and potential of differentiation into the chondrocyte, osteocyte and lipocytes lineages evaluated.
Results
The HPL materials had different protein composition, Calcium chloride activation removed most of fibrinogen. The same trends in growth factor content by ELISA in the four HPL fractions was found. BCEC morphology revealed increased shrinking in medium supplemented with SDPL, whereas the other conditions were not different from the control group. BCEC viability was similar in all groups to that using 10% FBS. WJMSC cells became bigger (100 μm) in the control group after 5 passages whereas cells morphology remained unchanged in the media supplemented with each HPL fraction the same as 20% FBS protein content. After 5 passages in all HPL-supplemented media, WJMSC showed higher proliferation rate, maintained their immunophenotypic characteristics, and exhibited enhanced capacity to differentiate into the osteocyte, chondrocyte, and adipocyte lineages.
Conclusion
All HPL fractions, apart SDPL, could be used as supplements of growth media for expanding BCEC and WJMSC ex vivo based on cell viability and doubling time. In addition, BCEC cell junction markers were maintained. WJMSC immunophenotypes and differentiation capacity were preserved over several passages in HPL-supplemented media and showed healthier morphology than when expanded in FBS-supplemented medium.