Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection
Background: Inflammation and fibrosis are the primary occurrences of chronic antibody-mediated rejection (CABMR) in kidney transplant patients. Fibroblasts are the primary cell type involved in allograft rejection and play a crucial role in the pathogenesis and progression of chronic antibody-mediat...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2024-12-01
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| Series: | Indian Journal of Transplantation |
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| Online Access: | https://journals.lww.com/10.4103/ijot.ijot_19_24 |
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| author | Mantabya Kumar Singh Mohit Kumar Rai Vikas Agarwal Narayan Prasad |
| author_facet | Mantabya Kumar Singh Mohit Kumar Rai Vikas Agarwal Narayan Prasad |
| author_sort | Mantabya Kumar Singh |
| collection | DOAJ |
| description | Background:
Inflammation and fibrosis are the primary occurrences of chronic antibody-mediated rejection (CABMR) in kidney transplant patients. Fibroblasts are the primary cell type involved in allograft rejection and play a crucial role in the pathogenesis and progression of chronic antibody-mediated rejection (CABMR). The in vitro study of the fibroblast is essential for comprehending biological processes and molecular reasons for CABMR and creating innovative treatment methods. However, establishing primary cultures from the kidney tissue is challenging.
Aim and Objective:
This protocol describes a simple and reproducible method for selective propagation and culture of primary human kidney fibroblasts from kidney cortex tissue. Techniques for their isolation and characterization are described in detail.
Material and Methods:
Primary kidney fibroblast cell culture was performed with a single core of fresh allograft biopsy tissue collected from the patients with CABMR. The biopsy tissue was collected in normal saline or phosphate buffer saline (PBS) and transported immediately on ice to the cell culture laboratory of the department.
Results:
An inverted microscope imaging shows the fibroblast cells during the first passage at 14th day, the cells were characterized by fingerprint monolayers when they were more than 80 % confluent. During the fourth passage at day 90, these cells took on the appearance of a long, elongated, spindle shaped. The immunofluorescence (IF) staining, shows the primary kidney fibroblast cells at the fourth passage were strongly positive for the fibroblast marker, Collagen I (fibrogenic cells), α-SMA (Resident fibroblast to active myofibroblasts conversion), and Vimentin (mesenchymal cells). On the other hand, the epithelial marker, cytokeratin-8, and E-cadherin were found to be very weak positive.
Conclusion:
This study provides an optimized, simple, and cost-effective method for primary fibroblast cultures from kidney tissue that may be reproducible easily at various laboratories and will provide a rich resource for future studies and research. |
| format | Article |
| id | doaj-art-a671d85919bf4e8ba1cd747ec700e767 |
| institution | Kabale University |
| issn | 2212-0017 2212-0025 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Indian Journal of Transplantation |
| spelling | doaj-art-a671d85919bf4e8ba1cd747ec700e7672025-01-07T06:12:53ZengWolters Kluwer Medknow PublicationsIndian Journal of Transplantation2212-00172212-00252024-12-0118443143510.4103/ijot.ijot_19_24Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant RejectionMantabya Kumar SinghMohit Kumar RaiVikas AgarwalNarayan PrasadBackground: Inflammation and fibrosis are the primary occurrences of chronic antibody-mediated rejection (CABMR) in kidney transplant patients. Fibroblasts are the primary cell type involved in allograft rejection and play a crucial role in the pathogenesis and progression of chronic antibody-mediated rejection (CABMR). The in vitro study of the fibroblast is essential for comprehending biological processes and molecular reasons for CABMR and creating innovative treatment methods. However, establishing primary cultures from the kidney tissue is challenging. Aim and Objective: This protocol describes a simple and reproducible method for selective propagation and culture of primary human kidney fibroblasts from kidney cortex tissue. Techniques for their isolation and characterization are described in detail. Material and Methods: Primary kidney fibroblast cell culture was performed with a single core of fresh allograft biopsy tissue collected from the patients with CABMR. The biopsy tissue was collected in normal saline or phosphate buffer saline (PBS) and transported immediately on ice to the cell culture laboratory of the department. Results: An inverted microscope imaging shows the fibroblast cells during the first passage at 14th day, the cells were characterized by fingerprint monolayers when they were more than 80 % confluent. During the fourth passage at day 90, these cells took on the appearance of a long, elongated, spindle shaped. The immunofluorescence (IF) staining, shows the primary kidney fibroblast cells at the fourth passage were strongly positive for the fibroblast marker, Collagen I (fibrogenic cells), α-SMA (Resident fibroblast to active myofibroblasts conversion), and Vimentin (mesenchymal cells). On the other hand, the epithelial marker, cytokeratin-8, and E-cadherin were found to be very weak positive. Conclusion: This study provides an optimized, simple, and cost-effective method for primary fibroblast cultures from kidney tissue that may be reproducible easily at various laboratories and will provide a rich resource for future studies and research.https://journals.lww.com/10.4103/ijot.ijot_19_24collagen iculture protocolcytokeratin-8e-cadherinenzymatic digestionimmunofluorescencekidney fibroblastvimentinα-smooth muscle actin |
| spellingShingle | Mantabya Kumar Singh Mohit Kumar Rai Vikas Agarwal Narayan Prasad Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection Indian Journal of Transplantation collagen i culture protocol cytokeratin-8 e-cadherin enzymatic digestion immunofluorescence kidney fibroblast vimentin α-smooth muscle actin |
| title | Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection |
| title_full | Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection |
| title_fullStr | Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection |
| title_full_unstemmed | Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection |
| title_short | Isolation, Culture, and Characterization of Primary Kidney Fibroblasts from Human Patients with Chronic Antibody-mediated Kidney Transplant Rejection |
| title_sort | isolation culture and characterization of primary kidney fibroblasts from human patients with chronic antibody mediated kidney transplant rejection |
| topic | collagen i culture protocol cytokeratin-8 e-cadherin enzymatic digestion immunofluorescence kidney fibroblast vimentin α-smooth muscle actin |
| url | https://journals.lww.com/10.4103/ijot.ijot_19_24 |
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