The preparation of lens epithelial explants has been adapted from methods originated in the laboratory of Dr. John McAvoy 9. This work is funded by the National Eye Institute, Intramural Research Program Z01-EY000238-22

Part 1: Removal of lenses
Materials and reagents:
<ol>
<li>Micro-dissecting scissors, curved, blunt tips, (such as.RS-5983, Roboz Surgical Instrument Co., Inc, Gaithersburg, MD); micro-dissecting tweezers, curved tip (such as Roboz #RS5137).</li>
<li>Suspension medium: Medium 199 containing 0.1% bovine serum albumin, 100 units/ml penicillin, 100&mu;g/ml streptomycin, 2.5 &mu;g/ml Amphotericin B. Reagents are available from Invitrogen, Carlsbad, CA.</li>
</ol>
Procedure:
<ol>
<li>Euthanize newborn rats (aged 2-4 days) in accordance with guidelines provided by the National Institutes of Health, Bethesda, MD.</li>
<li>Remove the eyelids with surgical scissors. Press gently with curved tweezers on opposite sides of the eye socket to force the eye to bulge outward. Make a small incision on the posterior side of the eye with the scissors. By pressing with tweezers against the side of the eye opposite the incision, the lens and a small amount of attached vitreous body can then be forced out through the rupture, allowing the lens to be picked up with curved tweezers. Care should be taken not to break the lens capsule.</li>
<li>Use the curved tweezers to transfer the lenses to a 60mm plastic tissue culture dish containing 5ml warm, sterile suspension medium.</li>
</ol>
Part 2: Microdissection of explants
Materials and reagents:
<ol>
<li>Micro Dissecting Tweezers, 0.1 mm tips, (such as Roboz RS-4976). We use Dumostar alloy because of the flexibility of the tip, but other steel alloys are also acceptable. Tweezers with tips thinner than 0.1mm are not recommended, as they tend to break or bend during this procedure.</li>
<li>Suspension medium (see Part 1)</li>
<li>Unsupplemented Ham&#x2019;s F12 medium or Medium 199 (Invitrogen, Carlsburg, CA)</li>
</ol>
Procedure:
<ol>
<li>The following steps should be carried out in a clean, draft-free environment using sterile dissecting tools and sterile medium.</li>
<li>Using a stereo-dissecting microscope, clean the lenses of any adhering tissue with the 0.1 mm tip tweezers and transfer them to a second 60mm culture dish containing 5ml sterile suspension medium (this step helps to reduce contamination). With the tweezers, transfer the desired number of lenses to a 35mm culture dish containing 5ml sterile, unsupplemented medium. (We often use Ham&#x2019;s F12 (Invitrogen) for this step, because the lower dye concentration makes dissection easier; however, Medium 199 is also acceptable. No antibiotics or other additions are needed during this step.) As many as 12 explants can be made in the center of a dish this size. However, a 35 mm dish should be used even if fewer explants are to be made, because the dissecting tools cannot be manipulated easily in a smaller dish. Transfer the dish containing the remaining lenses to a tissue culture incubator at 37&deg;C until they are needed.</li>
<li>Identify the posterior side of the lens. There are many ways to recognize this: 1) The posterior is rounder than the anterior, which is slightly flattened; 2) Newborn rat lenses form cold cataracts as they cool to room temperature during the dissection. If the cold cataract has not completely filled the lens, the posterior side is the side farthest from the opaque region. If the opacity has filled the lens, warming the dish to 37&deg;C for a few minutes will reverse it. The posterior side can be identified as the cataract reforms upon cooling. 3) Vestiges of the tunica vasculosa lentis may be visible on the posterior side. 4) The posterior suture may be visible on the posterior side. Identifying the posterior side correctly is essential, since this is where the capsule will be opened. Opening the lens on the anterior side will tear the epithelium.</li>
<li>Once the posterior side has been identified, turn it upward and grasp the lens in the left tweezers (for a right-handed worker). Then pinch the posterior capsule with the right tweezers to produce a small fold.</li>
<li>While holding the posterior capsule with the right tweezers, grasp the fold of capsule with the left tweezers and pull the two pairs of tweezers in opposite directions to make a small tear in the capsule.</li>
<li>While grasping the edge of the retracting capsule with the tweezers, pull it downward, first on one side, then on the other, pressing it into the plastic of the dish with the tweezers. Repeat this several times, moving all around the equator of the lens, until the capsule is firmly attached to the plate at many points. Holding the capsule in place with the left tweezers, gently rock the fiber mass with the right tweezers to break the attachment between the fiber cells and epithelial cells at the lens equator. Then push the fiber mass away, rolling it off the capsule/epithelium, which remains attached to the bottom of the dish. Continue this process with all the lenses in the dish. Remove and discard the fiber masses (or save them frozen as a source of lens proteins for other studies).</li>
</ol>
Part 3: Microdissection and culture of central explants
Materials and reagents:
<ol>
<li>Sterile disposable scalpel, #15 blade, (Cincinnati Surgical Co. Cincinnati, OH)</li>
<li>Sterile phosphate buffered saline with calcium and magnesium (Invitrogen, Carlsbad, CA)</li>
<li>Culture medium: Suspension medium containing 100ng/ml FGF-2 (Sigma-Aldrich, Inc. St Louis, MO).</li>
</ol>
Procedure:
<ol>
<li>Using a sterile scalpel, trim away the peripheral epithelium (PE), which contains cells in the early stages of differentiation (Figure 1A), leaving a central square approximately 2.0mm on a side, consisting of central epithelial cells only (CE) (Figure 1B).</li>
<li>Transfer the dish containing the central explants to a biosafety cabinet. Wash 3 times with sterile PBS containing calcium and magnesium and once with 1ml of fresh, equilibrated (37&deg;C, 5% CO2) suspension medium. These washes greatly reduce the likelihood of contamination.</li>
<li>Add 2mL of culture medium to induce differentiation1, and place the explants in a humidified, tissue culture incubator at 37&deg;C , 5% CO2. Culture periods may be as short as a few hours or extend as long as two to three weeks, depending on the parameter being studied. Medium should be changed every two to three days.</li>
</ol>
Part 4: Harvesting of explants for analysis of events associated with differentiation
1. Harvesting explants for analysis of protein or RNA 
	Materials:
	<ol>
 	<li>Microdissecting tweezers</li>
	<li>SDS Lysis Buffer or RNA-later</li>
	</ol>
	Procedure:
	<ol>
		<li>At the end of the incubation period, remove the culture medium.</li>
		<li>Using the stereo-dissecting microscope, gently loosen the edges of each explant.</li>
		<li>Lift each explant with the tweezers and transfer it to a tube containing about 100&mu;l SDS lysis buffer (for protein analysis) or RNA-later (for RNA analysis). Agitate the tip of the tweezers in the solution to ensure that the explant does not stick to the tweezers.</li>
	</ol>
2. Harvesting explants for immunofluorescence
Materials and reagents:
	<ol>
		<li>Phosphate buffered saline with calcium and magnesium (Invitrogen, Carlsbad, CA)</li>
		<li>Phosphate buffered saline (without calcium and magnesium) (Invitrogen, Carlsbad, CA)</li>
		<li>4% paraformaldehyde (Boston Bioproducts, Worcester, MA)</li>
		<li>Microdissecting tweezers</li>
		<li>Hydrophobic marking pen</li>
		<li>Glass microscope slides</li>
		<li>0.25% Triton X-100 in phosphate buffered saline.</li>
	</ol>
	Procedure:
	<ol>
		<li>Rinse explants briefly in PBS containing calcium and magnesium.</li>
		<li>Fix tissues by adding 4% paraformaldehyde for 30 minutes at room temperature.</li>
		<li>Remove the fixative and replace with phosphate buffered saline. Fixed explants become somewhat stiff, allowing them to be lifted and transferred to glass slides for immunostaining.</li>
		<li>Place a small drop of PBS (without calcium and magnesium) on the slide to help position the tissue and prevent curling. Using microdissecting tweezers, lift the explant and insert it into the drop on the slide. Without touching the explant, carefully remove the liquid with a paper wick to flatten the explant on the glass and let the tissue dry in air at room temperature for 3 to 5 minutes.</li>
	</ol>
<img src="/files/ftp_upload/1519/fig1.jpg" alt="Figure 1" /> Figure 1 A. The peripheral epithelium expresses differentiation-specific proteins. The explant shown was immunostained for N-cadherin immediately after microdissection. Expression is seen in a band of cells in the peripheral epithelium, indicating that cells in this region have begun to differentiate. B. The peripheral epithelium can be trimmed away to remove cells that express N-cadherin and other differentiation-specific proteins. The red annulus represents the location of cells that express N-cadherin, while the small square outlined in gray represents the quadrant of the epithelium shown in panel 1A. The peripheral epithelium can be removed by four scalpel cuts, leaving a central square that contains only cells that have not yet begun to differentiate.

<ol>
	<li>McAvoy, J. W., Chamberlain, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+(FGF)+induces+different+responses+in+lens+epithelial+cells+depending+on+its+concentration.">Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration.</a> Development. 107 (2), 221-228 (1989).</li><li>Campbell, M. T., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+of+fibre+differentiation+in+cultured+rat+lens+epithelium+under+the+influence+of+neural+retina-conditioned+medium.">Onset of fibre differentiation in cultured rat lens epithelium under the influence of neural retina-conditioned medium.</a> Exp Eye Res. 39 (1), 83-94 (1984).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+lens+epithelial+explants+induced+to+differentiate+into+fibres+by+fibroblast+growth+factor+(FGF).">Structural analysis of lens epithelial explants induced to differentiate into fibres by fibroblast growth factor (FGF).</a> Exp Eye Res. 49 (3), 479-494 (1989).</li><li>Golestaneh, N., Fan, J., Fariss, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+major+intrinsic+protein+(MIP)/aquaporin+0+expression+in+rat+lens+epithelia+explants+requires+fibroblast+growth+factor-induced+ERK+and+JNK+signaling.">Lens major intrinsic protein (MIP)/aquaporin 0 expression in rat lens epithelia explants requires fibroblast growth factor-induced ERK and JNK signaling.</a> J Biol Chem. 279 (30), 31813-31822 (2004).</li><li>Saravanamuthu, S. S., Gao, C. Y., Zelenka, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+lateral+induction+of+Jagged1+during+FGF-induced+lens+fiber+differentiation.">Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation.</a> Dev Biol. 332 (1), 166-176 (2009).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-induced+lens+cell+proliferation+and+differentiation+is+dependent+on+MAPK+(ERK1/2)+signalling.">FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.</a> Development. 128 (24), 5075-5084 (2001).</li><li>Dirks, R. P., Kraft, H. J., Van Genesen, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cooperation+between+two+silencers+creates+an+enhancer+element+that+controls+both+the+lens-preferred+and+the+differentiation+stage-specific+expression+of+the+rat+beta+B2-crystallin+gene.">The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.</a> Eur J Biochem. 239 (1), 23-32 (1996).</li><li>Yang, Y., Stopka, T., Golestaneh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+alphaA-crystallin+via+Pax6,+c-Maf,+CREB+and+a+broad+domain+of+lens-specific+chromatin.">Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.</a> The EMBO journal. 25 (10), 2107-2118 (2006).</li><li>McAvoy, J. W., T, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+retinas+promote+cell+division+and+fibre+differentiation+in+lens+epithelial+explants.">Neural retinas promote cell division and fibre differentiation in lens epithelial explants.</a> Curr Eye Res. 3 (6), 827-834 (1984).</li></ol>

The rat lens explant system has been successfully used by a number of laboratories to study terminal differentiation of lens epithelial cells to lens fibers 21,3,4,5. When exposed to FGF-2 at 100ng/ml the explants will begin to show changes in signaling within minutes 6, with changes in morphology and gene expression appearing sequentially over several days 3,4,5. The cultures remain viable for 2-3 weeks if care is taken to prevent contamination.
The central explants described in this protocol are especially useful for studying the sequence of events associated with differentiation, since they contain few if any cells that express differentiation markers before FGF-2 is added 1,5. The cells then differentiate synchronously, as a cohort, making it possible to follow the time course of signaling and transcriptional events associated with differentiation. Culturing explants for different lengths of time thus provides accurate temporal information about the sequence events. Specific inhibitors may be added to the culture medium to identify relevant signaling pathways. Explants may be used to analyze protein expression by SDS gel electrophoresis and immunoblotting or to analyze expression of specific mRNAs by RT-PCR. Protein yields range from 20-50 &mu;g/explant and RNA yield is approximately 200 &#x2013; 600 ng/explant, depending on the length of the culture period. We generally find that 5-6 explants per dish will provide sufficient protein or RNA for several assays. RNA from explants can also be used to prepare cDNA for assessing gene expression by microarray analysis, which can identify novel genes that may be critical for differentiation. Explants may also be transfected. Although transfection efficiency is generally low, it is sufficient for assaying reporter genes 4;7;8. Immunofluorescence microscopy of the explants provides a useful adjunct to biochemical methods by determining the subcellular location of proteins of interest. Thus, the preparation and culture of rat lens explants provides a powerful system for studying terminal lens differentiation in mammals, which can complement in vivo techniques, such as generation of transgenic and knock-out mice.

preparation and culture of rat lens epithelial explants for studying terminal differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Watch this Scientific Journal Video about Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation at JoVE.com

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

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12.6K 조회수.  National Eye Institute (NEI), National Institutes of Health (NIH). FGF - 2의 존재 양식 때 쥐 렌즈 상피 조직의 중심 지역의 Explants는 동기 구별. 이러한 문화의 Immunofluorescence 현미경은 유전자 발현 및 단말 차별과 연관된 신호 이벤트에 대한 소설 정보를 제공하실 수 있습니다.

비디오: Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Studying Protein Expression During Chicken Lens Development

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

Author Spotlight: Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

Preparation of Chicken Lens for Microinjection of an RCAS(A) Retrovirus

To begin, attach the borosilicate glass capillary to the rubber cushioned clips in the manual vertical micropipette puller. Set the heat temperature of the micropipette puller to 950 degrees Celsius and perform pre-pulling to obtain a thinner and softer glass capillary. Then set the heat temperature to 790 degrees Celsius and perform a secondary pulling to produce final micropipettes.Using a micropipette grinder, carefully sharpen the tip opening to an outer diameter of 11 micrometers. Then place the micropipettes in a sponge clamping pad inside of a glass jar. Incubate the fertilized chicken eggs until development stage 18 in a 37 degree Celsius humidified rocking incubator.Wipe down the working area thoroughly with 70%ethanol. After development, remove the eggs from the incubator. Spray the eggs with 70%ethanol and allow them to air dry.Then place the egg on an egg holder with the larger end facing upwards. Using a pair of sharp toothed forceps, carefully tap on the larger end of the eggshell to create a two centimeter diameter hole. Then remove the fragments of the egg shell.Place the egg on a dissecting microscope stage and locate the embryo. Next, use the fine dissecting forceps and scissors to cut off the amniotic membrane covering the top of the embryo. Then place a 60 millimeter Petri dish over the opening of the egg.