Dec 27, 2023
Non-Enzymatic Generation of Placenta Single Cells from Third Trimester Human Placenta
- Ameloko Joy1,2,
- Idowu Aimola1,2,
- Fomukong A Hanneda1,2,
- Reuben B. Samson1,2,
- Zeenat B Kudan1,2,
- Habiba H. Abubakar3,
- Musa Kana3,
- Benedict Anchang4
- 1Africa Center of Excellence for Neglected Tropical Diseases and Forensic Biotechnology, Ahmadu Bello University, Zaria Nigeria;
- 2Department of Biochemistry, Ahmadu Bello University Zaria, Nigeria;
- 3Kaduna Infant Development Birth Cohort Project Kaduna, Nigeria;
- 4National Institute of Environmental Health Sciences, Raleigh, North Carolina
Protocol Citation: Ameloko Joy, Idowu Aimola, Fomukong A Hanneda, Reuben B. Samson, Zeenat B Kudan, Habiba H. Abubakar, Musa Kana, Benedict Anchang 2023. Non-Enzymatic Generation of Placenta Single Cells from Third Trimester Human Placenta. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3b99l3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 13, 2023
Last Modified: December 27, 2023
Protocol Integer ID: 92360
Keywords: placenta single cell, single cell rna, viability for single cell rna, placental tissue with high yield, third trimester human placenta the placenta, placental tissue, third trimester human placenta, placenta, single cell, single cell suspension, fetal medicine, rna, multiple cell type, reduced viability of cell, complex organ with multiple cell type, cell, cell technology, sequencing, enzyme
Funders Acknowledgements:
Chan Zuckerberg Initiative
Grant ID: DAF2022-240134
Abstract
The placenta is a heterogeneous and complex organ with multiple cell types, posing a challenge for the field of maternal-fetal medicine to implement single-cell technologies for a deeper characterization of this essential organ. Several protocols use enzymes to digest the tissue and generate single cell suspension, but this approach has several shortcomings including the loss and reduced viability of cells. In this study, we describe a non-enzymatic approach to generate single cell suspension
from placental tissue with high yield and viability for single cell RNA
sequencing.
Attachments
Materials
| S/N | EQUIPMENT/CONSUMMABLES | COMPANY | |
| 1. | Styrofoam box with insulated container | Uline | |
| 2. | 1.5ml microcentrifuge tubes | Eppendorf | |
| 3. | Ice making machine | Kojak | |
| 4. | Weighing Balance | Halomedicals Systems Limited | |
| 5. | Gentle MACs Dissociator | Miltenyi Biotech | |
| 6. | Gentle MACs C- tubes | Miltenyi Biotech | |
| 7. | Countess III Chamber slides | Thermo Fisher Scientific | |
| 8. | Countess III Automated Cell counter | Thermo Fisher Scientific | |
| 9. | 100- 1000µl Micro pipette | Agros 240-21 Omega 8 | |
| 0.1- 10µl Micro pipette | Agros 240-21 Omega 8 | ||
| 10. | Refrigerated Micro Centrifuge | Eppendorf | |
| 11. | 100µl Pippete tips | Argos technologis | |
| 12. | 10µl Pippete tips | Argos technologis | |
| 13. | 1000µl Pippete tips |
Troubleshooting
Placenta Single Cell Preparation Protocol
10m 30s
Transform placenta sections into gentle MACs C-tube containing 3000 µL of MACs running buffer.
Set up a gentle MACS dissociator program to h-cord-01 for 553 rotation per round (rpr) at
00:00:30 .
30s
After the run, centrifuge at 300 x g for 00:10:00 at 40 °C .
10m
Remove the supernatant leaving 200 µL to resuspend pellet in. Transfer pipettes may be used
to remove supernatant after centrifugation to minimize loss of placenta cells.
10 µL of placental cells can be counted on a countess II cell counter or using trypan blue.