We would like to thank Alexandra Joyner for her support when this protocol was being established, Edwina McGlinn and Yi-cheng Chang for sharing retinoic acid. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

All the experiments should be carried out following the local governmental and institutional guidelines of ethical handling of laboratory animals.
1. Preparations Prior to the Day of Bone Culture
<ol>
	<li>Set up timed mouse matings to obtain fetuses and pups from embryonic day 14.5 (E14.5) and onward. 
	NOTE: The culture of long bones can be successfully applied to different mouse strains; in the present protocol, outbred Swiss Webster wild-type mice are used.</li>
	<li>Prepare dissection medium (adapted from Houston et al.15): dilute &#945;-minimum essential medium (&#945;-MEM) or Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) 1/13 in phosphate-buffered saline (PBS) and 2 mg/mL bovine serum albumin (BSA) and filter sterilize through a 0.22 &#181;m, 33 mm diameter syringe filter. Store aliquots at -20 &#176;C.</li>
	<li>The day before extraction of the fetuses, prepare the serum-free bone culture medium composed of DMEM containing 0.2% BSA, 0.5 mM L-glutamine, 40 U/mL penicillin/streptomycin, 0.05 mg/mL ascorbic acid, and 1 mM betaglycerophosphate. Filter sterilize through a 0.22 &#181;m, 33 mm diameter syringe filter and store at 4 &#176;C for up to 1 month.</li>
	<li>On the day of the culture and prior to mouse culling, prepare 24-well plates and 60 mm dishes with dissection medium and keep them ice cold. Prewarm the bone culture medium in a 37 &#176;C water bath. Spray 80% (v/v) ethanol on the tools (tweezers and small scissors) to be used for fetus handling. Transfer a binocular scope to a Class II biosafety cabinet.</li>
</ol>
2. Culture of Fetus and Newborn Tibia and Femur
<ol>
	<li>Cull the pregnant mouse through cervical dislocation at the desired gestational stage (ranging from E14.5 to E18.5). If newborn pups are used, remove them from the mother one by one and cull by decapitation.</li>
	<li>Place the mouse on her back and sterilize the abdominal region by spraying 80% (v/v) ethanol on its surface.</li>
	<li>Cut the skin and the abdominal muscle with small scissors to access the uterine horns.</li>
	<li>Extract the uterus from the abdominal cavity with the help of tweezers and small scissors, removing the mesometrium and cutting the base of the horns. Place the uterus in a 60 mm Petri dish with ice-cold dissection medium and keep the culture dish on ice during the whole procedure.</li>
	<li>Transfer the Petri dish with the uterus to the biosafety cabinet and work there from now on.</li>
	<li>Separate individual fetuses with scissors by cutting between the sacs.</li>
	<li>Transfer individual sac under a dissection stereomicroscope in a clean 60 mm dish with dissection medium and open them up with tweezers to separate the fetuses from the placentas and clean them from membranes. 
	NOTE: Work with one embryo at a time, while keeping the rest on ice.</li>
	<li>Decapitate the fetuses and transfer the body to a clean new 60 mm dish with a 1 mL cut sterile pipette.</li>
	<li>Remove the skin of the fetuses or pups with tweezers starting from the back and peeling it out till the toes.</li>
	<li>Separate the hindlimbs from the body by cutting with the tweezers close to the spine and transfer them to a clean dish with ice-cold dissection medium.</li>
	<li>Separate the tibia from the femur with tweezers by carefully introducing them between the surface cartilage of distal femur and proximal tibia.</li>
	<li>Remove the hip bones from the proximal femur and the calcaneus bone and the fibula from the tibia.</li>
	<li>Carefully remove the soft tissue from the femur and the tibia by nipping and pulling it off. 
	NOTE: It is important to remove as much soft tissue as possible, taking special care in removing the tissue that connects the two cartilage poles, but avoiding damaging the cartilage, the perichondrium, and the bones.</li>
	<li>Place the four bones (left and right tibias and femurs) in the first well of the 24-well plate with a plastic 1 mL sterile pipette. Alternatively, to compare the effect of different treatments on left and right limbs, place contralateral limbs in different wells. 
	NOTE: Extra care should be taken when the bones are transferred to the wells, as they can easily stick to the pipette.</li>
	<li>Proceed the same way with as many fetuses as necessary. 
	NOTE: Change the dish with the dissection medium as soon as it gets too clouded, as it is important to see clearly the dissected bones.</li>
	<li>When all the desired bones are transferred to the wells, remove the dissection medium with a plastic 1 mL sterile pipette and take extra care not to aspirate the bones.
	<ol>
		<li>Depending on the purpose of experiment, pictures can be taken of the bones before removing the dissection medium, as timepoint zero of the experiment. Take pictures with a microscope attached to a digital camera and annotate the exposure and scale used. To ensure easy and reliable measurements, take pictures with good contrast to distinguish the mineralized part.</li>
	</ol>
	</li>
	<li>Add 1 mL of culture medium to each well. If any treatment is intended on the bones (doxycycline, tamoxifen, growth factors, etc.), it should be added now.</li>
	<li>To observe the effect of growth inhibition in the culture conditions, treat the left tibias with retinoic acid (RA, 500 nM), while incubating the right tibias with an equivalent volume of vehicle (dimethyl sulfoxide [DMSO], final concentration 0.1%) as a control.</li>
	<li>Leave the bones to grow for two days or more in a cell culture incubator under standard cell culture conditions (at 37 &#176;C in a 5% CO2 incubator).</li>
	<li>To assess proliferation, add 5-ethynyl-2&#8217;-deoxyuridine (EdU) or 5-bromo-2&#8217;-deoxyuridine (BrdU) to the medium at a final concentration of 10 &#181;M 1&#8722;2 h before fixation. 
	NOTE: The stock concentration of EdU is 20 mM. 
	CAUTION: EdU and BrdU are thymidine analogues and can be toxic and mutagenic.</li>
	<li>Thaw 4% paraformaldehyde (PFA) and fix the bones by immersion in PFA in individual 2 mL tubes. 
	CAUTION: PFA is toxic and designated as a probable human carcinogen. Avoid breathing paraformaldehyde powder and vapors. EdU and BrdU are thymidine analogues and can be toxic and mutagenic.</li>
	<li>After a brief 10 min fixation in PFA at room temperature, transfer bones to PBS for picture acquisition at final timepoint. Then place bones back into PFA for overnight fixing at 4 &#176;C.</li>
	<li>After fixation bones can be processed for desired downstream applications.</li>
</ol>
3. Measurement and Analysis of the Full Length of the Bone and of the Mineralized Region
<ol>
	<li>Use an image editing software to measure the length of the bones, taking into account the scale of the image. Measure both total length of the bone and the mineralized region. Start the measurements from the first dark cells at one end until the last ones at the other end. 
	NOTE: The mineralized region is characterized by the darker color and is easily distinguished from the cartilage.</li>
	<li>To calculate the growth rate, defined as the average increase in length per day, divide the difference between the final length of the bone and the initial one by the number of days in culture.</li>
</ol>

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The authors have nothing to disclose.

Bone ex vivo culture methods have been used for some time to assess the biology of bone growth28, but have been seldom applied to murine long bones. With the development of imaging techniques, ex vivo bone culture offers an attractive way to study bone growth in real time in a setting closely resembling the in vivo conditions. In this scenario, it is important to define the conditions in which the growth of long bones is comparable to their growth in vivo.
In the presen...

Organ growth has to be tightly tuned to prevent the appearance of growth disorders, and involves the regulation of multiple cell types, molecular pathways and crosstalk among different parts of the body. Imaging techniques are essential to address the changes occurring over time in a growing embryo, both in normal conditions, as well as after a perturbation is induced in the system. Embryos with intrauterine development, such as the widely used rodent models, present an additional challenge for live imaging and drug treatment, which can be partially overcome by using ex vivo culture techniques. To successfully recapitulate the in vivo processes and obtain meaningful results, it becomes crucial to find the right culturing conditions for each organ or tissue.
Most bones of the mammalian skeleton grow through endochondral ossification, where the embryonic cartilage (composed of cells called chondrocytes) drives longitudinal growth and is gradually replaced by bone. This process happens at the growth plates, located at the end of the long bones, where three zones can be distinguished: resting, proliferative, and hypertrophic1,2. First, the round progenitor chondrocytes in the resting zone transition into the cycling columnar chondrocytes in the proliferative zone. During the next stage of differentiation, these chondrocytes become hypertrophic and start secreting type X collagen. Hypertrophic chondrocytes orchestrate the subsequent steps of ossification: they secrete key signaling molecules, such as connective tissue growth factor, bone morphogenetic proteins and Indian hedgehog, and direct the mineralization of the matrix, recruit blood vessels to the central part of the bone, and, upon apoptosis, allow osteoblasts (bone-forming cells) to invade the matrix to form the primary ossification center3,4. The mineralized matrix facilitates the penetration of blood vessels through which osteoblasts migrate to replace this degraded cartilage with a bone matrix5. Most osteoblasts invade the cartilage matrix from the perichondrium, a fibrous layer that wraps the cartilage6. Alternatively, a proportion of hypertrophic chondrocytes are able to survive and transdifferentiate to osteoblasts7,8,9. The final length of the bone is due to the accumulated growth of the transient cartilage, whose growth rate in turn depends on the number and size of the hypertrophic chondrocytes, and their matrix production10. Additionally, it was recently shown that the duration of the last hypertrophy phase correlates with the final length of the bone11. Therefore, tight regulation of the proliferation and differentiation of these cells is required to ensure proper bone size.
Despite of the substantial knowledge acquired over the years on the organization and development of the growth plates, most of these conclusions are based on the observation of fixed histological sections. Tissue sectioning provides valuable information about this process, but can be ridden with technical artifacts, so it cannot be always reliably used to estimate morphological or size changes between different stages. Additionally, as bone growth is a dynamic process, the static two-dimensional (2D) images offer a limited insight into the movement of the cells in the growth plate, while time-lapse imaging on live tissue could offer valuable information on the behavior of the chondrocytes in the growth plate.
All these limitations can be potentially resolved using ex vivo bone cultures. While bone culture protocols have been developed some time ago, they were limitedly applied to murine long bones. Most of the studies use chick bones due to the technical advantages offered by the chick model12,13. Organotypic cultures (air/liquid interface) were applied to chick embryonic femurs, which were maintained in culture for 10 days14. The sophisticated genetic tools available in mouse make this model very appealing to be used in ex vivo bone culture. The studies that used mice to look into bone growth worked mostly with metatarsal bones15, probably due to their small size and greater numbers obtained per embryo16. Although traditionally considered long bones, metatarsi enter senescence (characterized by reduced proliferation and involution of the growth plate17) earlier than other long bones in vivo, and therefore their continuous growth ex vivo does not really recapitulate the in vivo process. For the purposes of this article, we will use the term long bones for bones from the proximal and intermediate limb regions. Several previous studies used long murine bones, such as tibia, in ex vivo cultures and observed a substantial growth of the cartilage but little ossification18. We also used tibial cultures recently, mainly to study chondrocyte dynamics19. Other studies used femoral heads from young mice20 or only the distal part of the femur for culture21. Some more recent works successfully combine the ex vivo culture of full bones with time-lapse imaging to acquire three-dimensional (3D) movies of chondrocytes in living mouse tissue22,23. The authors managed to observe previously unnoticed events in the rearrangement of chondrocytes to the proliferative zone23 in a good example of the potential application of bone ex vivo culture. The alternative, i.e., analyzing static images, requires indirect and complex techniques. This was exemplified by a recent study assessing the importance of transversally-oriented clones for cartilage growth, where genetic tracing with multicolor reporter mouse strains coupled with mathematical modeling were used24. Therefore, ex vivo culture might help gain insight into dynamic processes in a faster and more straightforward way.
Here, we present a method for murine long bones culture, which can be combined with different molecular treatments and/or with time-lapse live imaging. This protocol adapts the methods used in previous reports15,18,25, but addresses some additional issues and focuses on long bones such as the tibia, rather than metatarsal bones. Finally, it explores the potential of using statistically powerful paired comparisons by culturing left and right bones separately in the presence of different substances.

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			<td height="21" style="height:21px;width:451px;">5-Ethynyl-2&#39;-deoxyuridine</td>
			<td style="width:132px;">Santa Cruz</td>
			<td style="width:119px;">CAS 61135-33-9</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-2&#8242;-deoxyuridine</td>
			<td style="width:132px;">Sigma</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:132px;">Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm TC-treated Center Well Organ Culture Dish, 20/Pack, 500/Case, Sterile</td>
			<td style="width:132px;">Falcon</td>
			<td>353037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adobe Photoshop</td>
			<td style="width:132px;">Adobe</td>
			<td>CS4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Ascorbic acid</td>
			<td style="width:132px;">Sigma</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Base unit for the scope</td>
			<td style="width:132px;">Zeiss</td>
			<td>435425-9100-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Betaglycerophosphate</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Binocular scope</td>
			<td style="width:132px;">Zeiss</td>
			<td style="width:119px;">STEMI-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Bovine Serum Albumin (BSA) fraction v</td>
			<td style="width:132px;">Roche/Sigma</td>
			<td style="width:119px;">10735086001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">DigiRetina 500 camera</td>
			<td style="width:132px;">Aunet</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Dissection kit</td>
			<td style="width:132px;">Cumper Robbins</td>
			<td style="width:119px;">PFS00034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">DMEM</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">11960044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">DMSO</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Eppendorf 2-mL tubes</td>
			<td style="width:132px;">Eppendorf</td>
			<td style="width:119px;">0030120094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Ethanol 96%</td>
			<td style="width:132px;">Merk</td>
			<td style="width:119px;">159010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Forceps Dumont#5 Inox08</td>
			<td style="width:132px;">Fine Science Tools</td>
			<td style="width:119px;">T05811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Heracell 150 CO2 incubator</td>
			<td style="width:132px;">Thermo Fisher</td>
			<td style="width:119px;">51026282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Minimum Essential Medium Eagle</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Multiwell 24 well</td>
			<td style="width:132px;">Falcon</td>
			<td style="width:119px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Paraformaldehyde</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Plastic pipettes 1mL Sterile Individually wrapped</td>
			<td style="width:132px;">Thermo</td>
			<td style="width:119px;">273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Syringe filter 0.2 um&#160;</td>
			<td style="width:132px;">Life Sciences</td>
			<td style="width:119px;">PN4612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Terumo syringe 20 mL</td>
			<td style="width:132px;">Terumo</td>
			<td style="width:119px;">DVR-5174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Tretinoin (retinoic acid)</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">PHR1187-3X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Trinocular scope</td>
			<td style="width:132px;">Aunet</td>
			<td style="width:119px;">AZS400T</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing and measuring fetal and newborn murine long bones

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Bone culture can be performed starting from different stages. In Figure 1A-D, a comparison between cultured tibia and freshly extracted ones at equivalent stages is shown. The first observation is that up to two days of culture the size achieved is comparable to the in vivo bone growth for both cartilage and mineralized bone (Figure 1A,B,D). Longer culture periods lead to bigger differences betwe...

Watch this Scientific Journal Video about Culturing and Measuring Fetal and Newborn Murine Long Bones at JoVE.com

Culturing and Measuring Fetal and Newborn Murine Long Bones

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

culturing-and-measuring-fetal-and-newborn-murine-long-bones

Research

JoVE Journal

Developmental Biology

8.1K Views.  Monash University. Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Video: Culturing and Measuring Fetal and Newborn Murine Long Bones

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...