A Novel Three-Dimensional Culture System Allows Prolonged Culture of Functional Human Granulosa Cells and Mimics the Ovarian Environment

Abstract

A novel three-dimensional culture system allows prolonged culture of functional human granulosa cells and mimics the ovarian environment AbstractThe development of techniques allowing the growth of primordial follicles to mature follicles in vitro has much potential for both reproductive medicine and developmental research. However, human primordial and preantral follicles fail to grow after isolation from the surrounding ovarian stroma. Granulosa cells (GCs), which normally undergo apoptosis after ovulation, contain a subpopulation of ovarian follicular cells remaining viable in vitro over prolonged time periods when cultured in the presence of leukaemia inhibiting factor (LIF). However, when cultured as monolayers, they progressively lose all their characteristics, such as follicle-stimulating hormone receptor (FSHR) and cytochrome P450-aromatase. Here, we describe a three-dimensional (3D) culture system containing type I collagen, which, together with LIF, allowed the survival and growth of a subpopulation of GCs isolated from mature ovarian follicles and supported them to proliferate into spherical structures exhibiting steroidogenic capacity, as demonstrated by P450-aromatase and 3-hydroxysteroid dehydrogenase. After transplantation into the ovaries of immuno-deficient mice, these cells became localized preferentially within antral follicles and the prolonged expression of FSHR was confirmed as well. With this optimization of the culture conditions an environment was created, which acts as a niche closely mimicking the development of early ovarian follicles in vitro. ENVIRONMENT (doi: 10.1089 This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2 ABSTRACTThe development of techniques allowing the growth of primordial follicles to mature follicles in vitro has much potential for both reproductive medicine and developmental research. However, human primordial and preantral follicles fail to grow after isolation from the surrounding ovarian stroma.Granulosa cells (GCs), which normally undergo apoptosis after ovulation, contain a subpopulation of ovarian follicular cells remaining viable in vitro over prolonged time periods when cultured in the presence of leukaemia inhibiting factor (LIF). However, when cultured as monolayers, they progressively lose all their characteristics, such as follicle-stimulating hormone receptor (FSHR) and cytochrome P450-aromatase. Here, we describe a three-dimensional (3D) culture system containing type I collagen, which, together with LIF, allowed the survival and growth of a subpopulation of GCs isolated from mature ovarian follicles and supported them to proliferate into spherical structures exhibiting steroidogenic capacity, as demonstrated by P450-aromatase and 3β-hydroxysteroid dehydrogenase. After transplantation into the ovaries of immuno-deficient mice, these cells became localized preferentially within antral follicles and the prolonged expressi...

Cells with Stem Cell Characteristics in Somatic Compartments of the Ovary

“…In the 3D model based on collagen type I in culture medium supplemented with LIF, Coll IV was found to surround patches of GCs containing the FSHR [60]. This is in accordance with the observation that in early antral follicles collagen type IV is localized specifically in the basal membrane [65], whereas in preovulatory follicles collagen type IV is also detected in more central layers of the granulosa [57].…”

“…It has now become possible to culture human GC over prolonged time periods in the presence of LIF, as set of experiments was designed to demonstrate that a significant subpopulation of human luteinizing GCs collected from mature ovarian follicles are able to maintain their functional characteristics over prolonged time period, when they are cultured in a 3D matrix made of collagen type I, and that they can become integrated into newly developing follicles after GC transplantation into the ovaries of immunoincompetent mice [60]. …”

“…The 3D culture system of GCs coated with collagen type I not only extended cellular survival in vitro but also allowed GCs to maintain many of their morphological and functional characteristics such as FSHR, LHR, and P450 aromatase [60]. The demonstration of both FSHR and Coll IV in GCs cultured in 3D suggested that this culture system mimics physiological ovarian follicular development (Figure 1).…”

Plasticity of granulosa cells: on the crossroad of stemness and transdifferentiation potential

“…The idea is to isolate stem cell population from GCs, enrich this population and transplant it to the patient. Kossowska-Tomaszczuk et al [40] developed a three-dimensional (3D) culture system which allowed the long-term survival, growth and enrichment of luteinizing GCs subpopulation isolated from mature ovarian follicles. The 3D system supported them to proliferate into spherical structures exhibiting steroidogenic capacity.…”

Selected molecular and physiological aspects of mammalian ovarian granulosa cells in primary culture

“…The most promising findings from human GC culture in support of their use in regeneration of endocrine functions were obtained in 2010. The three dimensional (3D) culture of GCs, which ensured longterm maintenance, growth, and also allowed enrichment with luteinizing GCs, was established by Kossowska et al (30). Moreover, this 3D culture system allowed GCs to grow into spherical structures that exhibit steroidogenic capability.…”