Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting
Purpose: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs...
| প্রধান লেখক: | , , , , |
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| বিন্যাস: | প্রবন্ধ |
| ভাষা: | English |
| প্রকাশিত: |
Elsevier
2022-05-01
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| মালা: | Chinese Journal of Traumatology |
| বিষয়গুলি: | |
| অনলাইন ব্যবহার করুন: | http://www.sciencedirect.com/science/article/pii/S1008127521001929 |
| _version_ | 1828365682676334592 |
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| author | Di Liu Jian-Hui Sun Hua-Cai Zhang Jian-Xin Jiang Ling Zeng |
| author_facet | Di Liu Jian-Hui Sun Hua-Cai Zhang Jian-Xin Jiang Ling Zeng |
| author_sort | Di Liu |
| collection | DOAJ |
| description | Purpose: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. Methods: Sprague-Dawley rats (3–4 weeks, 120–150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. Results: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12–16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. Conclusion: AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration. |
| first_indexed | 2024-04-14T05:34:12Z |
| format | Article |
| id | doaj.art-e5f8edabd10c4e02a6f801bba7e7aef4 |
| institution | Directory Open Access Journal |
| issn | 1008-1275 |
| language | English |
| last_indexed | 2024-04-14T05:34:12Z |
| publishDate | 2022-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Chinese Journal of Traumatology |
| spelling | doaj.art-e5f8edabd10c4e02a6f801bba7e7aef42022-12-22T02:09:42ZengElsevierChinese Journal of Traumatology1008-12752022-05-01253138144Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sortingDi Liu0Jian-Hui Sun1Hua-Cai Zhang2Jian-Xin Jiang3Ling Zeng4State Key Laboratory of Trauma, Burns and Combined Injuries, Daping Hospital, Army Medical University, Chongqing, 400042, ChinaMedical Center of Trauma and War Injury, Daping Hospital, Army Medical University, Chongqing, 400042, ChinaState Key Laboratory of Trauma, Burns and Combined Injuries, Daping Hospital, Army Medical University, Chongqing, 400042, ChinaState Key Laboratory of Trauma, Burns and Combined Injuries, Daping Hospital, Army Medical University, Chongqing, 400042, ChinaState Key Laboratory of Trauma, Burns and Combined Injuries, Daping Hospital, Army Medical University, Chongqing, 400042, China; Corresponding author.Purpose: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. Methods: Sprague-Dawley rats (3–4 weeks, 120–150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. Results: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12–16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. Conclusion: AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.http://www.sciencedirect.com/science/article/pii/S1008127521001929Alveolar epithelial cells type 1Alveolar epithelial cells type 2Three-dimensional cultureMagnetic activated cell sorting |
| spellingShingle | Di Liu Jian-Hui Sun Hua-Cai Zhang Jian-Xin Jiang Ling Zeng Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting Chinese Journal of Traumatology Alveolar epithelial cells type 1 Alveolar epithelial cells type 2 Three-dimensional culture Magnetic activated cell sorting |
| title | Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| title_full | Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| title_fullStr | Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| title_full_unstemmed | Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| title_short | Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| title_sort | continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting |
| topic | Alveolar epithelial cells type 1 Alveolar epithelial cells type 2 Three-dimensional culture Magnetic activated cell sorting |
| url | http://www.sciencedirect.com/science/article/pii/S1008127521001929 |
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