이 작품은 부분적으로 CSIC (I + D 프로젝트 C022)와 PEDECI​​BA에 의해 지원되었다. 저자는 조심스럽게이 프로젝트에 사용되는 모든 기니 돼지 표본을 제공하기 위해 MoFlo 셀 분류기, 그리고 메리 - 몬테비데오에 관한 그들의 관대 한 협업 세포 생물학 장치 (파스퇴르 연구소 드 몬테 비디오)에서 마리엘라 Bollati와 발렌 포로 감사드립니다. 그림 1 그림 2와 3, S. Karger AG, 바젤의 권한 표 1; BioMed 본부의 허가를 재현하고 그림 4와 5는 존 와일리 &amp; 선즈, Inc의 (에 의해 소유 저작권 허가를 복제하거나 적응했다 와일리 - 블랙웰, 2011).

1. 세포 현탁액의 준비 <ol><li> 이러한 IACUC 또는 동급으로 전문위원회의 권고에 따라 사용할 시편을 희생 (우루과이, 국가위원회의 동물 실험에 대한 [CNEA]). 우리의 경우, 펜 토바 비탈의 과량이 투여되었다. </li><li> 표준 승인 절차에 따라 고환을 해부하고 얼음처럼 차가운 DMEM 10 ML을 포함, 얼음 96mm 유리 페트리 접시에 배치 10 % 소 태아 혈청과 보충. </li><li> 투 니카 albuginea을 제거하고 2 사각형 조각으로 캡슐화가 고환을 잘라 - 양쪽에 3mm. </li><li> 그 조각은 다음 Medimachine, 조직이 뚫린 스테인리스 스크린과 금속 회 전자를 포함하는 일회용 기기 내부에 세분화 된 자동 기계 분쇄기로 처리됩니다. 50 μm의 일회용 단위 disaggregator에 스위치 및 프로세스에서 이러한 조각 중 1 - 5 이렇게하려면, 감기의 장소 1 ML은 DMEM 4를 보충<html> 제조업체의 간단한 지침에 따라 50 초합니다. </li><li> 바늘없이 5 ML의 주사기 - 3을 사용하여 세분화 장치에서 결과 세포 현탁액을 복구 할 수 있습니다. </li><li> 0.5 밀리리터 DMEM을 보충와 이전에 담가 50 μm의 나일론 메쉬를 통해 필터링합니다. </li><li> 젖은 25 μm의 나일론 메쉬, 얼음에 장소를 사용하여 다시 현탁액을 필터링합니다. </li><li> 네우 바우 챔버에서 셀을 1로 세포 농도를 조정 - 2 × 10 7 세포 / ML DMEM 배지로. 적어도 4 X 10 7 세포 / 고환 물질의 그램은 일반적으로 얻을 수 있습니다. </li><li> 마지막으로, 세포 응집을 방지하기 위해 0.2 %의 최종 농도 (2 - 나프톨 -6,8 - 디 술폰산, 칼륨 염) NDA를 추가합니다. </li><li> 선택 사항 : 제조업체의 지침에 따라, 동물 세포 시중에서 생존 키트와 함께 고환 세포 현탁액의 세포 생존을 확인합니다. </li></ol> 2. 유세포 분석 ve_content &quot;&gt; 우리의 propidium 요오드의 높은 농도 (PI)로 염색 세포의 분석을 위해 488 nm에서 방출하는 조정 일관된 아르곤 이온 레이저를 장착 한 벡톤 - 디킨슨 FACSVantage 흐름 cytometer에를 사용했습니다. (고정되지 않은에 PI 입구의 문제 스트레스 세포) 다른 9,10 해결되었습니다. <ol><li> PI 염색을위한 50 세포 현탁액에 μg / ML의 최종 농도에서 형광을 추가하고, 0시 10 분 ° 어둠 속에서 C에 대해 품어. </li><li> 레이저 전력은 100 mW의 및 26분의 575 밴드 패스 필터가 FL2에서 PI-방출 ​​형광을 수집하는 데 사용됩니다로 설정됩니다. </li><li> 우리는 70 μm의 노즐 FC 측정을 수행합니다. spermatocyte 인구를 정​​렬, 봉투로 3 분류 방울을 사용하여 일반-R 또는 Normal-C 모드에서 정렬을 설정합니다. 3시 샘플 및 수집 튜브를 유지 - 4 ° C에 냉동 장치를 사용하여. 초당 1,500 ~ 500의 속도로 세포를 분석 할 시료 차이를 조정합니다. </li><li> 분석하는 CellQuest 소프트웨어 (BD)를 사용하여다음 매개 변수 : 앞으로 분산 형 (FSC-H), 측면 산란 (SSC-H), 총 방출 형광 또는 펄스 면적 (FL2-A) 및 형광 방출 또는 펄스 폭 (FL2-W)의 기간. </li></ol> 또한, 우리는 5 μg / ML의 최종 농도에 중요한 염료 훽스트 33342을 고용하고 37 10 분 ° 어둠 속에서 C 동안 배양했다. 세포 분석은 MoFlo cytometer에 (DakoCytomation)를 통해 25 mW의에서 UV 여기 파장 레이저 운영을 탑재 해, 70 μm의 노즐을 사용하여 수행 하였다. 데이터 조작은 정상 V4.3 소프트웨어 (DakoCytomation)로 수행 하였다.

<ol>
	<li>Meistrich, M. L., Prescott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+and+nuclei+from+rodent+testes.">Separation of spermatogenic cells and nuclei from rodent testes.</a> Methods of Cell Biology. 15, 15-54 (1977).</li><li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation,+physical+characterization,+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse.">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li><li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li><li>Geisinger, A., Rodr&#237;guez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis.">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+velocity+sedimentation.">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry.">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., Santi&#241;aque, F., L&#243;pez, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells.">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li><li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+but+not+dead?+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide.">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., L&#243;pez-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis.">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li><li>Span&#242;, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+for+reproductive+biology.">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li></ol>

여기에 설명 된 최적화 방법은 효소 세제 치료를 피하고 좋은 세포 무결성 및 유형 비율을 유지, 매우 빠르고 재현성 방식으로 쥐 고환 조직에서 세포 현탁액의 준비를 할 수 있습니다. 절차 (15 분 스팬 고환 해부, 조직의 절단 및 가공 포함), 관련 최소한의 처리 및 효소 치료의 부재의 간결함은 주요 장점 중 일부입니다. 이러한 모든 원래의 세포 인구에 존재하는 물질의 대표 샘플이 필요할 때 중...

<table><tbody><tr><td>Medimachine system</td><td>BD</td><td>340587</td><td></td></tr><tr><td>Medicon unit (50 &amp;mu;m)</td><td>BD</td><td>340591</td><td></td></tr><tr><td>Filcon unit (50 &amp;mu;m)</td><td>BD</td><td>340603</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>430-2100</td><td></td></tr><tr><td>Fetal calf serum</td><td>PAA</td><td>A11-151</td><td></td></tr><tr><td>NDA</td><td>Chemos BmbH</td><td>277081</td><td></td></tr><tr><td>H&amp;#8211;chst 33342</td><td>Sigma-Aldrich</td><td>14533</td><td>Stock solution 5 mg/ml; used at a final concentration of 5 &amp;mu;g/ml</td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>287075</td><td>Stock solution 1 mg/ml; used at a final concentration of 50 &amp;mu;g/ml</td></tr></tbody></table>

simple and efficient technique for the preparation of testicular cell suspensions

포유류의 고환은 체세포 고환 세포와 생식 세포의 여러 단계를 포함하여 30 개 이상의 서로 다른 세포 유형을 포함 매우 복잡한 기관이다. 이 이질성은 정자 발달의 특정 단계에서 순수한 농축 세포 인구는 대부분의 분자 분석 1 필요에 따라, 포유류의 정자의 기초 연구에 관한 중요한 단점이다. 같은 Staput 2,3, 원심 elutriation 1 및 유동 세포 계측법 등 다양한 전략 (FC) 4,5는 차동 유전자 발현 연구를 활성화하기 위해 농축 또는 정제 고환 세포 인구를 얻기 위해 사용되어왔다. 이것은 세포가 가장 농축 / 정제 방법에 대한 정학 것을 요구된다. 이상적으로, 세포 현탁액은 원래의 조직을 대표하고, 가능한 세포와​​ 몇 multinucleates의 비중이 높은 때문에 -의 형성 경향이그리고 부족한 세포 대단히 짧은 시간 1 - 정액 상피 6,7의 세포 융합 성격. 이전 보고서는 독점적 기계적 방법에 의해 제조 고환 세포 현탁액은 트립신 사람 1보다 더 쉽게 응집 것을 입증했다. RNAses 및 / 또는 트립신 및 특정 스트림 애플리케이션에 바람직하지 않은 특정 고분자 분해에 콜라 리드와 같은 분해하는 효소 반면에, 효소 처리. 이상적인 프로세스는 mRNA가 같은 관심 고분자의 좋은 보존을 달성하기 위하여 가능한 한 짧아야하며 최소한의 조작을 포함해야한다. 단단한 조직에서 세포 현탁액의 준비를위한 현재 프로토콜은 일반적으로 시간이 걸리는 매우 연산자에 의존하고, 선택적으로 특정 세포 유형에게 1,8가 손상 될 수 있습니다. 여기에 제시 프로토콜과 높은 재현성 매우 간단한 기계 세분화의 이점을 결합유세포 분석 및 정렬 4 및 꿍꿍이 유전자 발현 연구 9에 사용할 수있는 좋은 품질의 세포 현탁액을 선도하는 효소 치료의 bsence.

여기에 설명 된 프로토콜을 준비 쥐의 고환에서 잘 분해 된 세포 현탁액의 예는 그림 1에 표시됩니다. 효소 치료 6,8에 이전에 설명한 기계 세분화 방법 2에 비해, 여기에 제시 한 (특히 다른 기계에 비해 훨씬 빠릅니다 적은 처리를 포함, (NOT 연산자에 의존) 쉽게 재현하고 부족한 세포 파편을 렌더링 방법)과 거의 multinucleates (광범위한 조직 조작 <s...

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

효소 세제를 피 설치류 자료에서 고환 세포 현탁액의 기계적 준비를위한 새로운 프로토콜이 설명되어 있습니다. 방법은 매우 간단하고 빠르고, 재현성 및 흐름 정렬 및 RNA 추출에 적합한 좋은 품질의 세포 현탁액을 렌더링합니다.

고환 세포 현탁액의 준비를 위해 간단하고 효율적인 기술

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

simple-efficient-technique-for-preparation-testicular-cell

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Biology

18.9K 조회수.  Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). 효소 세제를 피 설치류 자료에서 고환 세포 현탁액의 기계적 준비를위한 새로운 프로토콜이 설명되어 있습니다. 방법은 매우 간단하고 빠르고, 재현성 및 흐름 정렬 및 RNA 추출에 적합한 좋은 품질의 세포 현탁액을 렌더링합니다.

비디오: Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

기계 조직 해리 및 유동 세포 계측법에 의한 포유 동물 수컷 세포의 다중 종 정제에 대한 표준화 된 접근법

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

연구

발생학

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

표준 실험실 장비를 사용하여 마우스 고환에서 파키텐 정자 세포와 정자의 농축

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

쥐 고환의 세르 톨리 (Sertoli) 세포의 분리 및 세뇨관 세포

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

면역학 및 감염병학

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

살아있는, 그대로 조직에서 세포 분열의 분석을 위한 초파리 애벌레와 Pupal 고환의 준비

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

추가 세포 매트릭스 기반 및 추가 세포 매트릭스 -Murine 고환 오르가노이드의 추가 세포 매트릭스 없는 생성

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

보라색 흥분 세포 투과성 DNA 결합 염료를 사용하여 뮤린 정자 세포의 격리

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

생물학

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

장기 성인 마우스 SSC 라인의 유도를위한 문화 Spermatogonial 줄기 및 전구 세포의 일련 농축 (SSCs)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUT 속도의 침전을 사용하여 정자 형성 세포 유형의 분리

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

고환 세균 세포의 3차원 크로마틴 아키텍처를 보존하는 슬라이드 준비 방법

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...