Using <em>Ex Vivo</em> Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Abstract

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development. Video LinkThe video component of this article can be found at

Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads

“…The glucose concentration is crucial in the testis development process, especially for Sertoli cell differentiation from undifferentiated gonads [24]. A widely used DMEM (high glucose) supplemented with FBS or KSR was used for the culture medium in our experiments [9,29]. The success rates of the FBS-and KSR-supplemented groups were slightly different.…”

“…These results suggested that, in our own culture system, the proper biological pressure was provided through the maintaining the gonadal morphology by the addition of a semi-cylindrical piece of gel. A culture method using a small volume of culture medium suitable for RNA interference and inhibitory chemical additional experiments has also been reported [22,29,34]. The results of the gonadal culture including the filter method may be improved if the morphology is maintained with a suitable holding material.…”

“…Sufficient oxygen is supplied and nutrients are also supplied through micropores in the filter method. Culture in the gas phase is considered important to ensure an adequate oxygen supply [29,34]. The volume of the culture medium was adjusted to a position below the groove of the agarose gel block.…”

Stem cell-based therapies for fertility preservation in males: Current status and future prospects

“…Another culture method is the hanging drop technique, in which testicular tissue fragments are cultured in a small volume of medium placed on the lid of a culture plate. This method has been used to explore the effects of chemical treatments on human and mouse testes and to study fetal vascularization, morphogenesis, and organogenesis[ 120 , 121 ]. Pendergraft et al [ 122 ] used a hanging drop approach to produce a functional testicular organ system by coculture with adult SSCs.…”

Testicular organoids: a new model to study the testicular microenvironment in vitro?

“…An example of an organ-culture system is the hanging-drop method, where a fragment of testicular tissue is cultured within a small volume of medium placed on the lid of a culture dish. This method has been used to explore the effects of chemical treatments in human testis (Jorgensen et al, 2014) and to study human (Jorgensen et al, 2015) and murine (Potter and DeFalco, 2015) testicular development (Table II). Another organ-culture system is the air-liquid interface system, which consists of the culture of a small testicular tissue piece on a supportive stand and in simultaneous contact with the culture medium and the atmosphere.…”