Organotypic Culture

Freshney, R. Ian

doi:10.1002/0471747599.cac025

Cited by 4 publications

(5 citation statements)

References 71 publications

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“…As a rule in organ culture, tissues are generally placed at the interface between the liquid and gaseous phases, to facilitate gas exchange while retaining access to nutrients. Nonetheless, degeneration and necrosis in the central region frequently occur (Freshney, , Chapter 24). Raising the oxygen tension in the incubator could be helpful in certain cases, such as whole embryo culture for a short period of time, but not so in most other cases, due to the toxic effect of oxygen becoming apparent during longer periods (Ellis‐Hutchings & Carney, ; New, ; New, Coppola, & Terry, ).…”

Section: Discussionmentioning

confidence: 99%

Neonatal testis growth recreated in vitro by two‐dimensional organ spreading

Kojima

Nakamura

Komeya

et al. 2018

Biotech & Bioengineering

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Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

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Section: Discussionmentioning

confidence: 99%

Neonatal testis growth recreated in vitro by two‐dimensional organ spreading

Kojima

Nakamura

Komeya

et al. 2018

Biotech & Bioengineering

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“…The liquid-gas interphase method allows each tissue fragment easy access to oxygen [32]. Accordingly, Steinberger et al, seeking the beneficial effects of higher O 2 concentrations, tested 10% CO 2 þ 90% O 2 in their organ culture experiments and found that, although the penetration of O 2 into tissues was improved as shown by the smaller area of central necrosis in each tissue, the seminiferous tubules were more distorted and the numbers of spermatogonia and spermatocytes had decreased dramatically [8].…”

Section: Discussionmentioning

confidence: 99%

In Vitro Murine Spermatogenesis in an Organ Culture System1

Gohbara

Katagiri

Sato

et al. 2010

Biology of Reproduction

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Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr- and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

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“…Based on our present results, mainly focusing on the functional aspects of testis tissue, it was shown that adult tissues are vulnerable, compared to immature tissues, to the ex vivo conditions. Although mechanism is not clear, one plausible reason is that immature tissues are relatively resistant to hypoxic conditions, while adult tissues are not [20]. Thus, some studies used higher oxygen concentrations for culturing adult tissues.…”

Section: Discussionmentioning

confidence: 99%

“…However, simply raising the oxygen content in the culture incubator reportedly did not overcome this problem [9]. In fact, there is always a trade-off between the demand for oxygen and its toxicity to cells and tissues [20]. Testis tissue is no exception, and is certainly sensitive to hypoxic conditions.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues

et al. 2015

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Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

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Organotypic Culture

Cited by 4 publications

References 71 publications

Neonatal testis growth recreated in vitro by two‐dimensional organ spreading

Neonatal testis growth recreated in vitro by two‐dimensional organ spreading

In Vitro Murine Spermatogenesis in an Organ Culture System1

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues

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