Name: Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing
Uploaded: 2023-05-12T14:30:00
Description: Watch this Scientific Journal Video about Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing at JoVE.com

mouse cardiac progenitor cell isolation, digestion, and freezing

एकल-कोशिका संकल्प पर माउस कार्डियक पूर्वज कोशिकाओं से नाभिक अलगाव

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise to different subtypes of cardiac cells, including myocardial, fibroblast, epicardial, and endocardial cells4,5.&#160;Single-cell genomics is emerging as a key method for studying heart development and assessing the impact of cellular heterogeneity in health and disease6. The development of multi-omics methods for the simultaneous measurement of different parameters and the expansion of computational pipelines has facilitated the discovery of cell types and subtypes in the normal and diseased heart6. This article describes a reliable single-nucleus isolation protocol for frozen cardiac progenitor cells obtained from mouse embryos that is compatible with downstream snRNA-seq and snATAC-seq (as well as snRNA-seq and snATAC-seq combined)7,8,9.
ATAC-seq is a robust method that allows the identification of regulatory open chromatin regions and the positioning of nucleosomes10,11. This information is used to draw conclusions about the location, identity, and activity of transcription factors. The activity of chromatin factors, including remodelers, as well as the transcriptional activity of RNA polymerase, can, thus, be analyzed since the method is highly sensitive for measuring quantitative changes in chromatin structure1,2. Thus, ATAC-seq provides a robust and impartial approach to uncovering the mechanisms controlling transcriptional regulation in a specific cell type. ATAC-seq protocols have also been validated to measure chromatin accessibility in single cells, revealing variability in the chromatin architecture within cell populations10,12,13.
Although there have been notable advances in the field of single cells in recent years, the main difficulty is the processing of the fresh samples needed to perform these experiments14. To circumvent this difficulty, various tests have been carried out with the aim of conducting analyses such as snRNA-seq and snATAC-seq with frozen cardiac tissue or cells15,16.
Several platforms have been used to analyze single-cell genomics data17. The widely used platforms for single-cell gene expression and ATAC profiling are platforms for multiple microfluidic droplet encapsulation17. As these platforms use microfluidic chambers, debris or aggregates can clog the system, resulting in non-usable data. Thus, the success of single-cell studies depends on the accurate isolation of individual cells/nuclei.
The protocol presented here uses a similar approach to recent studies using snRNA-seq and snATAC-seq to understand congenital heart defects18,19,20,21,22,23. This procedure utilizes the enzymatic dissociation of freshly microdissected cardiac tissue followed by the cryopreservation of mouse cardiac progenitor cells. After thawing, the viable cells are purified and processed for nuclear isolation. In this work, this protocol was successfully used to obtain snRNA-seq and snATAC-seq data from the same nuclear preparation of mouse cardiac progenitor cells.

लेखक स्पॉटलाइट: एकल-सेल रिज़ॉल्यूशन पर एपिजेनोम और जीन अभिव्यक्ति प्रोफाइलिंग के लिए माउस कार्डियक पूर्वज कोशिकाओं से नाभिक अलगाव  

एकल-कोशिका संकल्प पर एपिजीनोम और जीन अभिव्यक्ति प्रोफाइलिंग के लिए माउस कार्डियक पूर्वज कोशिकाओं से नाभिक अलगाव

The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches such as single nuclei RNA-seq and single nuclei ataxic are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been a significant progress in the field in single cell for recent years, the main difficulty is processing free samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defects.This protocol describes the steps to follow for successfully isolating high quality nuclei from frozen cardiac cell suspension obtained from mouse embryos. And our protocol is compatible with downstream single nuclei RNA-seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research.

Watch this Scientific Journal Video about Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing at JoVE.com

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing  

माउस कार्डियक पूर्वज कोशिका अलगाव, पाचन, और ठंड

दो से छह महीने की गर्भवती मादा माउस से विच्छेदित भ्रूण को 1% भ्रूण गोजातीय सीरम के साथ ठंडे पूर्ण माध्यम में रखकर शुरू करें। 5एक्स आवर्धन के तहत भ्रूण से एंडोमेट्रियल ऊतक, प्लेसेंटा, और जर्दी बोरी को हटाने के बाद, भ्रूण के हृदय क्षेत्र से कार्डियक पूर्वजों को बल का उपयोग करके विच्छेदित करें। 1.5 मिलीलीटर माइक्रो सेंट्रीफ्यूज ट्यूब में पांच माउस भ्रूण से विच्छेदित सभी हृदय क्षेत्रों को खींचें और उन्हें एक झूलती बाल्टी में सेंट्रीफ्यूज करें, जिसे चार डिग्री सेल्सियस तक पूर्व-ठंडा किया जाए।सतह पर तैरनेवाला को हटा दें, और ऊतक को एक मिलीलीटर ऊतक संस्कृति ग्रेड पीबीएस से धो लें। ट्यूबों को चार डिग्री सेल्सियस पर एक मिनट के लिए 300 जी पर सेंट्रीफ्यूज करें। फिर, सतह पर तैरने वाले को हटा दें, और एक मिलीलीटर पीबीएस के साथ दो वॉश दोहराएं।एक बार हो जाने के बाद, पीबीएस को 0.05% ट्रिप्सिन-ईडीटीए से बदलें। सेंट्रीफ्यूज करें और 0.05% ट्रिप्सिन-ईडीटीए के साथ दो वॉश दोहराएं। ऊतक को पचाने के लिए, 0.05% ट्रिप्सिन-ईडीटीए के 50 माइक्रोलीटर जोड़ें और 37 डिग्री सेल्सियस पर 10 मिनट के लिए गर्म करें।इसके बाद, एक व्यापक-छिद्र फ़िल्टर टिप का उपयोग करके निलंबन को ऊपर और नीचे करके पांच मिनट के बाद एक सौम्य यांत्रिक पृथक्करण लागू करें। विघटित कोशिकाओं में 350 माइक्रोलीटर पूर्ण माध्यम जोड़कर ट्रिप्सिन पाचन गतिविधि को निष्क्रिय करें। सेल निलंबन को 40 माइक्रोमीटर सेल स्ट्रेनर के माध्यम से पारित करें।ताजा-अलग सेल निलंबन को केंद्रीकृत करके कोशिकाओं को ठंड के लिए तैयार करें। सतह पर तैरनेवाला को सावधानी से हटा दें और एक मिलीलीटर ठंडा ठंड माध्यम में गोली को फिर से निलंबित करें। सेल सस्पेंशन को प्री-कूल्ड क्रायो शीशी में स्थानांतरित करें, और क्रायो शीशी को प्री-कूल्ड फ्रीजिंग कंटेनर में रखें।अनुक्रमण प्रयोगों के लिए तरल नाइट्रोजन में स्थानांतरित करने से पहले ठंड कंटेनर को शून्य से 80 डिग्री सेल्सियस नीचे चार से आठ घंटे तक रखें।

mouse-cardiac-progenitor-cell-isolation-digestion-and-freezing

Research

JoVE Journal

Developmental Biology

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Begin by placing the embryos dissected from the euthanized two to six-month-old pregnant female mouse in a cold complete medium with 1%fetal bovine serum. After removing the endometrial tissue, placenta, and yolk sack from the embryo under 5X magnification, dissect the cardiac progenitors from the embryonic heart region using forceps. Pull all the heart regions dissected from five mouse embryos in a 1.5 milliliter micro centrifuge tube and centrifuge them in a swinging bucket, pre-chilled to four degrees Celsius.Remove the supernatant, and wash the tissue with one milliliter of tissue culture grade PBS. Centrifuge the tubes at 300G for one minute at four degrees celsius. Then, remove the supernatant, and repeat two washes with one milliliter PBS.Once done, replace the PBS with 0.05%Trypsin-EDTA. Centrifuge and repeat two washes with 0.05%Trypsin-EDTA. To digest the tissue, add 50 microliters of 0.05%Trypsin-EDTA and heat for 10 minutes at 37 degrees Celsius.Next, apply a gentle mechanical dissociation after five minutes by aspirating the suspension up and down using a wide-orifice filter tip. Inactivate the Trypsin digestion activity by adding 350 microliters of complete medium to the dissociated cells. Pass the cell suspension through a 40 micrometer cell strainer.Prepare the cells for freezing by centrifuging the freshly-dissociated cell suspension. Carefully remove the supernatant and resuspend the pellet in one milliliter of chilled freezing medium. Transfer the cell suspension into a pre-cooled cryo vial, and place the cryo vial in a pre-cooled freezing container.Place the freezing container at minus 80 degrees Celsius for four to eight hours before transferring it to liquid nitrogen for sequencing experiments.

Embryonic Heart Cell Thawing and Removal of Dead Cells

Begin by placing the frozen dissociated embryonic heart cell suspension containing cryo vials in a 37 degrees Celsius water bath for one to two minutes. Add one milliliter of complete medium, pre warmed at 37 degrees Celsius to the thawed suspension and mix it three times with a pipette. Then transfer the suspension to a 15 milliliter conical tube, containing 10 millimeters of warm complete medium.Pass the entire cell suspension over a 30 micrometer strainer and rinse the strainer with one to two milliliters of warm, complete medium. Collect the flow through in the same conical tube. Next, centrifuge the tubes at 300 G for five minutes.After removing the supernatant, wash the pellet with PBS and transfer the cell suspension into a 1.5 milliliter micro tube. Centrifuge the cell suspension and add 100 microliters of the provided magnetic microbeads to the pelleted cells. Homogenize the suspension five times using a wide boar pipette tip before 15 minutes of incubation at room temperature.In the meantime, rinse the magnetic separation column with 500 microliters of binding buffer. Once the incubation is complete, dilute the microbead cell suspension mixture using 500 microliters of binding buffer. Next, add 0.6 milliliters of the cell suspension to the magnetic separation column.Collect the live cell effluent in a 15 milliliter centrifuge tube. Next, rinse the 1.5 milliliter micro tube that contained the cell suspension using one milliliter of binding buffer. After rinsing, transfer the binding buffer from the 1.5 milliliter micro tube to the column and collect the effluent in the same 15 milliliter tube.Repeat the rinsing of the 1.5 milliliter tube using one milliliter of binding buffer. After centrifuging the effluent at 300 G for five minutes, remove the supernatant without disrupting the cell pellet. Add one milliliter of the PBS BSA solution and gently mix by pipetting using a wide orifice pipette tip.Then transfer the cell suspension into a 1.5 milliliter micro tube. Again, centrifuge the cell suspension and remove the supernatant without disrupting the cell pellet. Repeat the two washes of one milliliter of PBS BSA.After removing the one milliliter of PBS BSA, resuspend the cells in 100 microliters of PBS BSA solution. Mix it by pipetting 10 times. Determine the concentration by performing a trypan blue assay to ensure a cell count of more than or equal to 100, 000 cells and perform a fluorescence based assessment for the viability of the cells.The additional step of sorting and removing the dead cells after thawing allowed the procurement of a cleaner cell suspension with significantly higher cell viability and improved the quality of the starting sample. For more accuracy, two different counting methods were used to quantify the cells and nuclei, including trypan blue and the fluorescence based assessment of the viability. In this protocol, it was observed that the cell viability passed from 80 to 90%before nuclei isolation to less than 5%after nuclei isolation.

भ्रूण हृदय कोशिका पिघलना और मृत कोशिकाओं को हटाना

Nuclei Isolation from Cardiac Progenitor Cells

To begin, clean the working place and materials with 70%ethanol and RNase removing solution. Freshly prepare lysis buffer, lysis dilution buffer, 0.1 x lysis buffer, and wash buffer and maintain the buffers on ice. Pre-chill swinging bucket centrifuges to four degrees Celsius.Centrifuge the cardiac progenitor cell suspension in a swinging bucket centrifuge. Gently remove all the supernatant without touching the bottom of the tube. Add 100 microliters of chilled 0.1 x lysis buffer to the cell suspension, and mix it by pipetting 10 times.After five minutes of incubation on ice, add one milliliter of chilled wash buffer and mix it. After centrifuging the suspension, remove the supernatant and repeat two more washes with chilled wash buffer. Prepare the diluted nuclei buffer.After resuspending the nuclei in the nuclei buffer, place the nuclei stock solution on ice. When the quality of the isolated nuclei was assessed by the evaluation of the nuclear membrane morphology, the isolated nuclei appeared smooth and uniformly round under Bright-field microscopy indicating an effective isolation process.