Bio-AlteR® is a 3D primary cell culture of seminiferous tubules enabling ex vivo spermatogenesis, bridging the gap between in vitro to the in vivo techniques.
- This model allows the study of both spermatogenesis mitotic and meiotic phases, as well as the first steps of spermiogenesis.
- This model preserves the original 3D structure of the seminiferous tubules, and the Sertoli cells remain polarized which is crucial for the differentiation of germ cells.
- The blood testis barrier is maintained, which allows the study of drug permeation across the barrier.
Cells are cultured in a bicameral chamber. This system allows the study of the impact of compounds (substances, drugs or molecules) added into the basal compartment on both the blood testis barrier integrity and on differentiating germ cells. The basal compartment mimics the blood compartment whereas the apical compartment reproduces the seminiferous tubules lumen environment. The cell culture media can be analyzed to identify product release and explore biomarkers produced as a result of toxicity.
Bio-AlteR® keeps blood testis barrier functional throughout the culture period.
After 12 days of a culture of seminiferous tubules from 20-day-old rats:
Tight junction protein: Occludin in green, Gap junction protein Connexin-43 in red.
Tight and gap junctions build the « niche » for spermatogenesis.
The development of the meiotic step in testes of pubertal rats is very similar in vivo and in the BioAlteR ® model
|Age of rats||Leptotene||Zygotene||Pachytene||Diplotene|
|In vivo||23 days||18%||15,7%||65,1%||1,2%|
|In vivo||42 days||3,7%||12,5%||67,8%||16%|
|In vivo||100 days||2,3%||8,1%||83,1%||6,5%|
|Ex vivo(BioAlteR ®)||ulture of 23-days old rats (Day 16)||4,3%||14%||60%||21,7%|
(Geoffroy-Siraudin C, Perrard MH, Chaspoul F, Lanteaume A, Gallice P, Durand P, Guichaoua M.R. Validation of a rat seminiferous tubule culture system for studying toxicant impact on meiosis: effect of the hexavalent chromium exposure. Toxicol Sci, 2010, 116, 286-96.)
BioAlteR ® was used to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture.
BioAlteR ® was able to reliably reproduce testicular toxicity reported in vivo for these four compounds.
This pilot study demonstrates the use of this ex-vivo organotypic spermatogenic culture system to model in vivo testicular toxicity in response to compounds with diverse mechanisms of testicular toxicity and to decipher their mechanisms/modes of action.
|Compounds||BioAlteR ® Findings|
|1,3 DNB (6 & 60µM)||Sertoli cell injury with TEER disruption at both concentrations
Loss of pachytene spermatocytes and round spermatides within the 1st week (6µM)
Degenerescence of all meiotic cells within 1st week at 60µM
|MAA (0.5 & 2.5 mM)||Sertoli cell injury with concentration-responsive TEER disruption and Cx-43 expression
Dramatic decrease of pachytene spermatocytes, secondary spermatocytes and spermatids
|BPA (5 & 50µM)||Sertoli cell injury with concentration-responsive TEER disruption and Cx-43 expression
Disruption of meiosis (no progression to pachytene spermatocyte stage at 50µM)
|Lindane 5 & 30µM)||Sertoli cell gap junctions disruption with concentration-responsive disruption of Cx-43 within
1st week and mild reduction in TEER
Apoptosis of pachytene spermatocytes (loss of pachytene spermatocytes and spermatids)
Goldstein KM, Seyler DE, Durand P, Perrard MH, Baker TK. Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants. Reprod Toxicol. 2016, 60:92-103.
Trans-epithelial electric resistance (TEER) measurement ( blood testis barrier (BTB) functionality )
TEER measurement is a non-invasive technique that can be applied to determine the functionality of BTB.
Tights (and Gap) junctions between Sertoli cells are the main composant of the blood testis barrier which is essential for spermatogenesis.
Thanks to Bio-AlteR® model that keep BTB intact and functional, Kallistem is the only one Company that offers a validated rat TEER assay in the presence of the germ cells.
This standard screening assay is performed in first-line to give a quick answer about the potential toxicity of compounds to be tested. It allows the screening of a larger number of molecules compared to the other complementary assays.
TEER is regularly measured throughout the 21 days of Bio-AlteR® culture.
TEER measurement can be used to determine the functionality and the integrity of the Blood Testis Barrier in an easily quantifiable method.
Monitoring of blood testis barrier evolution throughout cell culture duration with a voltohmmeter.
Example of TEER measurement
Effect of Dinitrobenzene (DNB) on TEER in Bio-AlteR® (rat)
1,3-DNB disrupted dramatically BTB in a dose and time dependent manner
For the toxic compounds, Kallistem has validated several complementary assays to determine the mechanism of action involved in the testicular toxicity (non-exhaustive list: depending on the study, numerous options are discussed and offered to the customer).
I- Modification of cell number population
Cellular viability of somatic and germ cells
Quantification of the 6 main cell populations (flow cytometric analysis)
II- mRNA quantification (RT-qPCR analysis)
Blood Testicular Barrier markers
Testis cell population (Sertoli cells and germ cells)
III- Endocrine disruptors detection and MOA study
Investigation of hormone –dependent signalling pathways
(Androgens, estrogens, FSH, thyroid hormones,...expression analysis)
Example : Bio-Alter® model has demonstrated that drinking water quality have an impact on spermatogenesis.
Gene expression of Androgen Receptor (AR) and Estrogen Receptors (Era and ERb):
A clear endocrine disruption effect was observed with drinking water sample 3 (strong decrease of AR and ERb gene expression).
Tap water: 1 and 2
Mineral water in plastic bottle: 3 and 4
Species available with Bio-AlteR® model