Usage of the Cell Dissociation Technique in Reproductive Studies; Study on Mechanical Cell Dissociation in Male Mice in Maturity Course
Document Type : Original Article
Authors
1 Department of Basic Science, Faculty of Veterinary Medicine University of Tehran, Tehran, Iran
2 Department of Biology, Faculty of Biology Science, Kavian Institute of higher Education, Mashhad, Iran
3 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
Abstract
Cell dissociation techniques are one of the main methods for separating different cells from various tissues. These techniques consist of two main approaches: chemical and mechanical separation. Each technique has its own advantages and disadvantages, and the most suitable method should be selected. In the chemical method, speci c enzymes must be used. In the mechanical method, cells are separated by mechanical force. Normally, in most research studies, the chemical method using speci c enzymes is more commonly applied. In this study, 15 mice were used during the maturity phase and were grown under standard conditions. These mice were operated on after 32 days, and the testes were extracted. The cells were dissociated using the mechanical method. Finally, the samples were stained with Giemsa and examined. The advantages of these techniques include the separation of undamaged cells, which were observed in both methods. However, the main focus of this paper was mechanical cell dissociation, which did not damage the primary cells. Moreover, this method can be used for chromatin studies by counting each cell type and separating them to investigate testicular functions. Therefore, using mechanical cell dissociation for the seminiferous tubule wall could be a useful and economical option as a low-cost method for comparative studies involving cells in testicular tissue. The ndings suggest that mechanical dissociation provides an efficient and low-cost method for isolating intact testicular cells, making it suitable for histological and functional studies of seminiferous tubules.
Keywords
References
[1] Schneider F, Redmann K, Wistuba J, Schlatt S,
Kliesch S, Neuhaus N. Comparison of enzymatic
digestion and mechanical dissociation of human
testicular tissues. Fertility and sterility. 2015 Aug
1;104(2):302-11.
[2] Bellve AR, Millette CF, Bhatnagar YM, O'Brien
DA. Dissociation of the mouse testis and charac-
terization of isolated spermatogenic cells. Jour-
nal of Histochemistry & Cytochemistry. 1977
Jul;25(7):480-94.
[3] Zhang R, Sun J, Zou K. Advances in isolation
methods for spermatogonial stem cells. Stem cell
reviews and reports. 2016 Feb;12(1):15-25.
[4] Lima AC, Jung M, Rusch J, Usmani A, Lopes AM,
Conrad DF. A standardized approach for multi-
species puri cation of mammalian male germ cells
by mechanical tissue dissociation and ow cytome-
try. Journal of visualized experiments: JoVE. 2017
Jul 12(125):55913.
[5] Lima AC, Jung M, Rusch J, Usmani A, Lopes AM,
Conrad DF. Multispecies puri cation of testicu-
lar germ cells. Biology of reproduction. 2016 Oct
1;95(4):85-1.
[6] Tepperman K, Morris G, Essien F, Heywood SM.
A mechanical dissociation method for preparation
of muscle cell cultures. Journal of Cellular Physi-
ology. 1975 Dec;86(3):561-5.
[7] Montanari M, Burattini S, Ciacci C, Ambrogini P,
Carloni S, Balduini W, Lopez D, Panza G, Papa
S, Canonico B. Automated-mechanical procedure
Avicenna Veterinary Research/ Vol. 1, No. 3, Summer 2025 13
compared to gentle enzymatic tissue dissociation
in cell function studies. Biomolecules. 2022 May
14;12(5):701.
[8] Goldberg S. Mechanical/physical methods of cell
distribution and tissue homogenization. InPro-
teomic Pro ling: Methods and Protocols 2015 Jan
1 (pp. 1-20). New York, NY: Springer New York.
[9] Schol J, Ambrosio L, Tamagawa S, Joyce
K, Ruiz-Fernandez C, Nomura A, Sakai D.
Enzymatic chemonucleolysis for lumbar disc
herniation-an assessment of historical and contem-
porary efficacy and safety: a systematic review
and meta-analysis. Scienti c Reports. 2024 Jun
4;14(1):12846.
[10] Worthington CC. Worthington enzyme manual:
enzymes and related biochemicals: Worthington
Biochemical Corporation; 1988.
[11] Wu S, Zhou X, Jin Z, Cheng H. Collagenases and
their inhibitors: a review. Collagen and Leather.
2023;5(1):19.
[12] Wu S, Zhou X, Xu K, Cheng H. The role of extrac-
tion method to collagen substrates in enzymolysis
of type I collagenase. International Journal of Bi-
ological Macromolecules. 2024;283:138086.
[13] Shajib MS, Futrega K, Jacob Klein T, Craw-
ford RW, Doran MR. Collagenase treatment ap-
pears to improve cartilage tissue integration but
damage to collagen networks is likely perma-
nent. Journal of Tissue Engineering. 2022; 13:
20417314221074207.
[14] Alipour H, Raz A, Zakeri S, Djadid ND. Ther-
apeutic applications of collagenase (metallopro-
teases): A review. Asian Paci c Journal of Tropi-
cal Biomedicine. 2016;6(11):975-81.
[15] Shetab-Boushehri SM-H, Shetab-Boushehri SV. A
Simple Efficient Inexpensive Device for Separation
of Trypsinized Cells from Tissue Fragments. Stem
Cell Reviews and Reports. 2024;20(3):855-6.
[16] Broadley C, Menzies G, Bramley T, Watson E.
Isolation of cell populations from the mare corpus
luteum: comparison of mechanical and collagenase
dissociation. Reproduction. 1994;102(1):7-15.
[17] Canavan HE, Cheng X, Graham DJ, Ratner BD,
Castner DG. A plasma-deposited surface for cell
sheet engineering: advantages over mechanical
dissociation of cells. Plasma Processes and Poly-
mers. 2006;3(6-7):516-23.
[18] Abbasi-Malati Z, Roushandeh AM, Kuwahara Y,
Roudkenar MH. Mesenchymal stem cells on hori-
zon: a new arsenal of therapeutic agents. Stem
Cell Reviews and Reports. 2018;14:484-99.
[19] Othman GQ, Mustafa TA, Ismael HD, Ali SS, Ab-
dullah HH, Abdullah B. A comparative investiga-
tion applying testicular ne needle aspiration cy-
tology and open testicular biopsy histology for the
diagnosis of azoospermia and severe oligospermia.
Archives of Razi Institute. 2023;78(4):1343.
[20] Tsoulou TK, Tsakmakidis IA, Tzika E, Kritsepi-
Konstantinou M. Spermatozoa under the micro-
scope: Moving beyond Giemsa. Veterinary Clini-
cal Pathology. 2020;49(4).
[1] Schneider F, Redmann K, Wistuba J, Schlatt S,
Kliesch S, Neuhaus N. Comparison of enzymatic
digestion and mechanical dissociation of human
testicular tissues. Fertility and sterility. 2015 Aug
1;104(2):302-11.
[2] Bellve AR, Millette CF, Bhatnagar YM, O'Brien
DA. Dissociation of the mouse testis and charac-
terization of isolated spermatogenic cells. Jour-
nal of Histochemistry & Cytochemistry. 1977
Jul;25(7):480-94.
[3] Zhang R, Sun J, Zou K. Advances in isolation
methods for spermatogonial stem cells. Stem cell
reviews and reports. 2016 Feb;12(1):15-25.
[4] Lima AC, Jung M, Rusch J, Usmani A, Lopes AM,
Conrad DF. A standardized approach for multi-
species puri cation of mammalian male germ cells
by mechanical tissue dissociation and ow cytome-
try. Journal of visualized experiments: JoVE. 2017
Jul 12(125):55913.
[5] Lima AC, Jung M, Rusch J, Usmani A, Lopes AM,
Conrad DF. Multispecies puri cation of testicu-
lar germ cells. Biology of reproduction. 2016 Oct
1;95(4):85-1.
[6] Tepperman K, Morris G, Essien F, Heywood SM.
A mechanical dissociation method for preparation
of muscle cell cultures. Journal of Cellular Physi-
ology. 1975 Dec;86(3):561-5.
[7] Montanari M, Burattini S, Ciacci C, Ambrogini P,
Carloni S, Balduini W, Lopez D, Panza G, Papa
S, Canonico B. Automated-mechanical procedure
Avicenna Veterinary Research/ Vol. 1, No. 3, Summer 2025 13
compared to gentle enzymatic tissue dissociation
in cell function studies. Biomolecules. 2022 May
14;12(5):701.
[8] Goldberg S. Mechanical/physical methods of cell
distribution and tissue homogenization. InPro-
teomic Pro ling: Methods and Protocols 2015 Jan
1 (pp. 1-20). New York, NY: Springer New York.
[9] Schol J, Ambrosio L, Tamagawa S, Joyce
K, Ruiz-Fernandez C, Nomura A, Sakai D.
Enzymatic chemonucleolysis for lumbar disc
herniation-an assessment of historical and contem-
porary efficacy and safety: a systematic review
and meta-analysis. Scienti c Reports. 2024 Jun
4;14(1):12846.
[10] Worthington CC. Worthington enzyme manual:
enzymes and related biochemicals: Worthington
Biochemical Corporation; 1988.
[11] Wu S, Zhou X, Jin Z, Cheng H. Collagenases and
their inhibitors: a review. Collagen and Leather.
2023;5(1):19.
[12] Wu S, Zhou X, Xu K, Cheng H. The role of extrac-
tion method to collagen substrates in enzymolysis
of type I collagenase. International Journal of Bi-
ological Macromolecules. 2024;283:138086.
[13] Shajib MS, Futrega K, Jacob Klein T, Craw-
ford RW, Doran MR. Collagenase treatment ap-
pears to improve cartilage tissue integration but
damage to collagen networks is likely perma-
nent. Journal of Tissue Engineering. 2022; 13:
20417314221074207.
[14] Alipour H, Raz A, Zakeri S, Djadid ND. Ther-
apeutic applications of collagenase (metallopro-
teases): A review. Asian Paci c Journal of Tropi-
cal Biomedicine. 2016;6(11):975-81.
[15] Shetab-Boushehri SM-H, Shetab-Boushehri SV. A
Simple Efficient Inexpensive Device for Separation
of Trypsinized Cells from Tissue Fragments. Stem
Cell Reviews and Reports. 2024;20(3):855-6.
[16] Broadley C, Menzies G, Bramley T, Watson E.
Isolation of cell populations from the mare corpus
luteum: comparison of mechanical and collagenase
dissociation. Reproduction. 1994;102(1):7-15.
[17] Canavan HE, Cheng X, Graham DJ, Ratner BD,
Castner DG. A plasma-deposited surface for cell
sheet engineering: advantages over mechanical
dissociation of cells. Plasma Processes and Poly-
mers. 2006;3(6-7):516-23.
[18] Abbasi-Malati Z, Roushandeh AM, Kuwahara Y,
Roudkenar MH. Mesenchymal stem cells on hori-
zon: a new arsenal of therapeutic agents. Stem
Cell Reviews and Reports. 2018;14:484-99.
[19] Othman GQ, Mustafa TA, Ismael HD, Ali SS, Ab-
dullah HH, Abdullah B. A comparative investiga-
tion applying testicular ne needle aspiration cy-
tology and open testicular biopsy histology for the
diagnosis of azoospermia and severe oligospermia.
Archives of Razi Institute. 2023;78(4):1343.
[20] Tsoulou TK, Tsakmakidis IA, Tzika E, Kritsepi-
Konstantinou M. Spermatozoa under the micro-
scope: Moving beyond Giemsa. Veterinary Clini-
cal Pathology. 2020;49(4).
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