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Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

Komeya M, Kimura H, Nakamura H, Yokonishi T, Sato T, Kojima K, Hayashi K, Katagiri K, Yamanaka H, Sanjo H, Yao M, Kamimura S, Inoue K, Ogonuki N, Ogura A, Fujii T, Ogawa T - Sci Rep (2016)

Bottom Line: Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective.Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months.The produced sperm were functional to generate healthy offspring with micro-insemination.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama, Kanagawa 236-0004, Japan.

ABSTRACT
In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

No MeSH data available.


Related in: MedlinePlus

Micro-insemination experiments and testosterone production.(A) Testis tissue cultured for 41 days included many spermatids (upper panels), which was dissected for harvesting haploid cells. ROSI and ICSI procedures produced healthy offspring (middle panels). Genomic PCR demonstrated that 7 out of 14 offspring carried the GFP gene, revealing their origin to be the transgenic sire, heterozygous for Acr-Gfp (lower left panel). They normally matured and were observed on 346 dpp (lower right panel). (B) Testis tissue cultured for 185 days was used for microinsemination (upper panels). Both ROSI and ICSI produced healthy offspring (middle panels). Genomic PCR demonstrated that all 11 offspring carried the GFP gene, revealing their origin to be the transgenic sire, homozygous for Acr-Gfp. Lanes P and N were loaded with a positive-control sample (genomic DNA of Acr-Gfp mouse) and a negative-control sample (genomic DNA of ICR mouse), respectively (lower left panel). The pups normally matured and were observed on 66 dpp (lower right panel). (C) Testosterone production by two cultured testis tissues, sample 1 & 2, was measured at 4 and 5 weeks by collecting medium flowing out of the device for 24 hours each. LH was added to the culture medium on the next day and medium samples were collected in the same way for testosterone measurement. (D) Testis tissue cultured for 120 days produced testosterone and responded to LH at 1 hour and onward. Scale bar: 1 cm (A,B middle panels), 500 μm (A upper left panel, B upper left & middle panels), 100 μm (A upper middle & right panels), 50 μm (B upper right panel).
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f4: Micro-insemination experiments and testosterone production.(A) Testis tissue cultured for 41 days included many spermatids (upper panels), which was dissected for harvesting haploid cells. ROSI and ICSI procedures produced healthy offspring (middle panels). Genomic PCR demonstrated that 7 out of 14 offspring carried the GFP gene, revealing their origin to be the transgenic sire, heterozygous for Acr-Gfp (lower left panel). They normally matured and were observed on 346 dpp (lower right panel). (B) Testis tissue cultured for 185 days was used for microinsemination (upper panels). Both ROSI and ICSI produced healthy offspring (middle panels). Genomic PCR demonstrated that all 11 offspring carried the GFP gene, revealing their origin to be the transgenic sire, homozygous for Acr-Gfp. Lanes P and N were loaded with a positive-control sample (genomic DNA of Acr-Gfp mouse) and a negative-control sample (genomic DNA of ICR mouse), respectively (lower left panel). The pups normally matured and were observed on 66 dpp (lower right panel). (C) Testosterone production by two cultured testis tissues, sample 1 & 2, was measured at 4 and 5 weeks by collecting medium flowing out of the device for 24 hours each. LH was added to the culture medium on the next day and medium samples were collected in the same way for testosterone measurement. (D) Testis tissue cultured for 120 days produced testosterone and responded to LH at 1 hour and onward. Scale bar: 1 cm (A,B middle panels), 500 μm (A upper left panel, B upper left & middle panels), 100 μm (A upper middle & right panels), 50 μm (B upper right panel).

Mentions: Then, fertility-competence of the haploid cells produced in the MF device was evaluated by performing micro-insemination experiments. Firstly, haploid cells were harvested from 2 tissue samples which were cultured for 41 days. Round spermatid injection (ROSI) and intracytoplasmic sperm injection (ICSI) procedures were successfully performed to produce 9 and 5 offspring, respectively. They all grew normally (Fig. 4A, Supplementary Table S1). Next, haploid cells were collected from testis tissue cultured for 185 days which contained numerous spermatids and sperm. Micro-insemination procedures produced 6 and 5 healthy offspring with ROSI and ICSI, respectively. They grew healthily and matured into adults (Fig. 4B, Supplementary Table S2). These results demonstrated that spermatogenesis in microfluidic devices can produce haploid cells that are competent male gametes.


Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

Komeya M, Kimura H, Nakamura H, Yokonishi T, Sato T, Kojima K, Hayashi K, Katagiri K, Yamanaka H, Sanjo H, Yao M, Kamimura S, Inoue K, Ogonuki N, Ogura A, Fujii T, Ogawa T - Sci Rep (2016)

Micro-insemination experiments and testosterone production.(A) Testis tissue cultured for 41 days included many spermatids (upper panels), which was dissected for harvesting haploid cells. ROSI and ICSI procedures produced healthy offspring (middle panels). Genomic PCR demonstrated that 7 out of 14 offspring carried the GFP gene, revealing their origin to be the transgenic sire, heterozygous for Acr-Gfp (lower left panel). They normally matured and were observed on 346 dpp (lower right panel). (B) Testis tissue cultured for 185 days was used for microinsemination (upper panels). Both ROSI and ICSI produced healthy offspring (middle panels). Genomic PCR demonstrated that all 11 offspring carried the GFP gene, revealing their origin to be the transgenic sire, homozygous for Acr-Gfp. Lanes P and N were loaded with a positive-control sample (genomic DNA of Acr-Gfp mouse) and a negative-control sample (genomic DNA of ICR mouse), respectively (lower left panel). The pups normally matured and were observed on 66 dpp (lower right panel). (C) Testosterone production by two cultured testis tissues, sample 1 & 2, was measured at 4 and 5 weeks by collecting medium flowing out of the device for 24 hours each. LH was added to the culture medium on the next day and medium samples were collected in the same way for testosterone measurement. (D) Testis tissue cultured for 120 days produced testosterone and responded to LH at 1 hour and onward. Scale bar: 1 cm (A,B middle panels), 500 μm (A upper left panel, B upper left & middle panels), 100 μm (A upper middle & right panels), 50 μm (B upper right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759809&req=5

f4: Micro-insemination experiments and testosterone production.(A) Testis tissue cultured for 41 days included many spermatids (upper panels), which was dissected for harvesting haploid cells. ROSI and ICSI procedures produced healthy offspring (middle panels). Genomic PCR demonstrated that 7 out of 14 offspring carried the GFP gene, revealing their origin to be the transgenic sire, heterozygous for Acr-Gfp (lower left panel). They normally matured and were observed on 346 dpp (lower right panel). (B) Testis tissue cultured for 185 days was used for microinsemination (upper panels). Both ROSI and ICSI produced healthy offspring (middle panels). Genomic PCR demonstrated that all 11 offspring carried the GFP gene, revealing their origin to be the transgenic sire, homozygous for Acr-Gfp. Lanes P and N were loaded with a positive-control sample (genomic DNA of Acr-Gfp mouse) and a negative-control sample (genomic DNA of ICR mouse), respectively (lower left panel). The pups normally matured and were observed on 66 dpp (lower right panel). (C) Testosterone production by two cultured testis tissues, sample 1 & 2, was measured at 4 and 5 weeks by collecting medium flowing out of the device for 24 hours each. LH was added to the culture medium on the next day and medium samples were collected in the same way for testosterone measurement. (D) Testis tissue cultured for 120 days produced testosterone and responded to LH at 1 hour and onward. Scale bar: 1 cm (A,B middle panels), 500 μm (A upper left panel, B upper left & middle panels), 100 μm (A upper middle & right panels), 50 μm (B upper right panel).
Mentions: Then, fertility-competence of the haploid cells produced in the MF device was evaluated by performing micro-insemination experiments. Firstly, haploid cells were harvested from 2 tissue samples which were cultured for 41 days. Round spermatid injection (ROSI) and intracytoplasmic sperm injection (ICSI) procedures were successfully performed to produce 9 and 5 offspring, respectively. They all grew normally (Fig. 4A, Supplementary Table S1). Next, haploid cells were collected from testis tissue cultured for 185 days which contained numerous spermatids and sperm. Micro-insemination procedures produced 6 and 5 healthy offspring with ROSI and ICSI, respectively. They grew healthily and matured into adults (Fig. 4B, Supplementary Table S2). These results demonstrated that spermatogenesis in microfluidic devices can produce haploid cells that are competent male gametes.

Bottom Line: Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective.Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months.The produced sperm were functional to generate healthy offspring with micro-insemination.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama, Kanagawa 236-0004, Japan.

ABSTRACT
In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

No MeSH data available.


Related in: MedlinePlus