This work was supported by the Ovarian Cancer Research Fund [grant LT/UIC/01.2011], the UIC Center for Clinical and Translational Science, the UIC Cancer Center, and the National Institutes of Health grant C06RR15482.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by AAALAC under approved animal care protocols by the Animal Care and Use Committee at UIC. Animals for this project are housed in barrier rooms in the Biological Resources Laboratory (BRL) at the University of Illinois at Chicago. The BRL has a fully veterinary staff that monitors animals at least twice daily and provides advice on animal care.

1. Ovarian dissection and tissue isolation
<ol>
	<li>Prepare a 0.5% (w/v) solution of sodium alginate, prepare from a modified version of the protocol described previously5, using sterile PBS and heat to 37&deg;C. Mix by inversion or rocking, but do not vortex. Draw the alginate into a 1 ml syringe with a 25 G needle and maintain at 37&deg;C.</li>
	<li>Prepare 10mM sterile CaCl2 solution for crosslinking the alginate upon tissue encapsulation and warm to 37&deg;C.</li>
	<li>Prepare 1mL of culture medium (&alpha;MEM + 5 units penicillin and 5 &mu;g/ml streptomycin) per well in a 24 well plate for each experiment. Include various drugs, peptides, or viruses in the culture medium. For transfection prior to organ encapsulation, incubate tissue with Lipofectamine 2000 and plasmid DNA for four hours at 37 &deg;C.</li>
	<li>Dissect the organs from the animal, remove any fat or residual tissue, gently pull off the bursa membrane surrounding the ovary, and cut the ovary or oviduct into four pieces by bisecting the organ. Animals were euthanized by CO2 asphyxiation followed by cervical dislocation. For the oviduct, gently uncoil the tissue by pulling apart the connecting membranes and extending the ends of the tube in opposite directions using forceps before cutting.</li>
	<li>Place a single alginate droplet (~1-3 &mu;L) onto a sterilized mesh fiber.</li>
	<li>Using sterile forceps, move a piece of organ into the alginate droplet and center the droplet using forceps or a sterile needle.</li>
	<li>Invert the droplet containing the organ piece (organoid) by grasping the mesh fiber with sterile forceps and turning it upside down to allow gravity to force the alginate into a hanging drop.</li>
	<li>Tap the forceps on the edge of a 10cm petri dish containing 10mM CaCl2 solution so that the drop falls into solution.</li>
	<li>Incubate for 2 minutes, remove with sterile spoon containing pores to release extra 10mM CaCl2, and transfer into the culture medium. Up to four alginate gels can be transferred into a single well of a 24 well plate containing the culture medium. Culture for a defined time period.</li>
</ol>
2. Degradation of alginate and collection of cells
<ol>
	<li>For the solubilization of the alginate gel, incubate the cultures in alginate lyase (3.4 mg/ml) dissolved in Leibovitz's L-15 media at 37&deg;C for 30 minutes or until gel is completely dissolved.</li>
	<li>Remove the tissue from the alginate lyase.</li>
	<li>For collection of the OSE, incubate the organoid with 500 units of collagenase in Leibovitz's L-15 media for 1 hour8. Vortex in 2 second pulses (2 seconds vortex and 2 seconds rest) on medium power for 1 minute, followed by 1 second pulses (1 second vortex and 1 second rest) for 1 minute. Let organ pieces fall to bottom of microcentrifuge tube and pipette supernatant into fresh sterile microcentrifuge tube. Centrifuge 3 minutes at 3000 rpm to pellet ovarian surface epithelium.</li>
	<li>For collection of the tubal epithelium, cut the tube lengthwise and place on tissue culture plastic in DMEM with 5 units penicillin and 5 &mu;g/ml streptomycin and 4% (v/v) FBS. Over 3-5 days, cells will "crawl" out of the tubal stroma and grow on tissue culture plastic9.</li>
</ol>
3. Fixation of alginate encapsulated organ cultures for paraffin sections
<ol>
	<li>Collect the alginate encapsulated bead from the culture medium at selected time point.</li>
	<li>Recrosslink the alginate to stiffen the gel by dropping the culture into sterile 10mM CaCl2 solution for two minutes.</li>
	<li>Fix the alginate encapsulated organ in 2% paraformaldehyde containing 50 mM sucrose, 10 mM CaCl2 and 50 mM sodium cacodylate to ensure that the gel stays solid and fully encapsulates tissue during fixation.</li>
	<li>Following tissue processing, embed the entire alginate-encapsulated organ culture piece into paraffin and section with a microtome to generate 5 &mu;m sections.</li>
	<li>The alginate will be dissolved and removed from the slide if performing immunohistochemistry due to heat based antigen retrieval. The alginate will remain and stain for eosin under standing hematoxylin and eosin staining.</li>
</ol>
4. Representative Results:
An example of a three-dimensional organ culture of the ovary and oviduct is shown in Figure 1. Ovaries can be cut into different size pieces, and OSE proliferate and migrate to repair the surface that was wounded from the incision with the scalpel. During this process, the correct orientation of the OSE relative to the underlying stroma is maintained, with the OSE encapsulating the ovary. As shown in Figure 2, the OSE of ovaries cut in half encapsulate the ovary more completely as compared to ovaries cut into quarters or eighths. The wound repair of the ovarian surface can be studied in the context of the entire ovary and may shed light on how ovulation and surface repair induces ovarian surface transformation.
The endpoint of the experiment depends on the information that is required. Tissue architecture, cellular morphology, and specific cellular growth rates are easily monitored using paraffin sections. For example, a single layer of ovarian surface epithelium is observed after culturing in serum-free &alpha;MEM for 7 days (Figure 3A) while addition of 5 &mu;g/ml insulin for 7 days induces hyperproliferation of the ovarian surface epithelium and formation of multiple layers of cells (Figure 3B). By adding bromodeoxyuridine to the cultures at a final concentration of 10 &mu;M 24 hr prior to fixation, proliferation of cells can be measured (inset 3A and 3B). For cultures maintained in serum-free &alpha;MEM, approximately 3-5% of OSE undergo proliferation during the 24 hr labeling period (inset 3A), while a larger percentage of granulosa cells proliferate. Addition of insulin to the culture media increases the percentage of proliferating OSE to approximately 30% of total OSE (inset 3B). Immunofluorescence of green fluorescent protein (GFP) can be visualized through the alginate gel with standard microscopy following cDNA transfection (Figure 3C). While alginate organ culture provides for growth and analysis of ovarian surface epithelium and primary follicles, it should be noted that the mature, secondary follicles fail to survive in this culture system (Figure 3D). Culture of individual follicles to maturity within an alginate hydrogel matrix has been described elsewhere10.
Alterations in protein or RNA can be analyzed from the entire organ or from specific cell types using enzymatic degradation (Figure 4). Treatment of cultured ovarian organoids with collagenase leaves the underlying stroma intact (Figure 4C) while allowing for enrichment of OSE in the cell pellet obtained following treatment, although some stromal cells are also isolated (Figure 4D).
<img alt="Figure 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> 
Figure 1. Mouse ovary three-dimensional culture system used to propagate OSE after wounding. A) Under the microscope, the bursa and the surrounding fat pad were removed. B) Intact ovary (O) with oviductal tube (T) and fat pad (F). C) Ovary with bursa and fat pad removed (left) and uncoiled oviduct (right). D) Ovarian or oviductal organoids were placed into an alginate droplet. E) Alginate encased organoids were dropped into a CaCl2 solution crosslinking the alginate to form a gel. F) Alginate encapsulated ovarian organoid. G) Alginate encapsulated oviductal organoid. H) Alginate encapsulated organoids incubated in culture medium.
<img alt="Figure 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> 
Figure 2. Surface area regenerated by ovarian surface measured by cytokeratin 8. Encapsulation of ovarian organoids by the OSE after being cut into eighths, quarters, and halves, cultured in BSA medium, and measured by immunohistochemical analysis using cytokeratin 8 (CK8) antibody.
<img alt="Figure 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> 
Figure 3. Alginate culture system allows passage of growth factors and transfection of cDNA into the OSE, but does not support rapid growth and maturation of follicles. A) Ovarian organoid cultured for 7 days in basal medium analyzed for CK8 expression, which marks the OSE. Inset, serial section stained for BrdU incorporation. B) Addition of insulin to the culture media induces hyperplasia of the OSE. Inset, serial section stained for BrdU incorporation. C) Ovarian organoid transfected with cDNA to express GFP. D) Alginate culture system supports growth of primordial follicles (red arrowhead), but not mature secondary follicles (black arrow).
<img alt="Figure 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> 
Figure 4. OSE can be isolated for analysis following culture. A) Organoid cultured in medium for up to 2 weeks. B) Encapsulated organoid is treated with alginate lyase to leave organoid intact. Ovary is stained with CK8 to mark OSE. C) Organoid is treated with collagenase to separate OSE from underlying stroma. Remaining tissue piece was stained with CK8 to highlight removal of the ovarian surface. D) Collected cells following treatment are enriched for OSE as stained for CK8 (red). Cells are counterstained with DAPI to mark nuclei.

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev. 22, 255-288 (2001).</li><li>Choi, J. H., Wong, A. S., Huang, H. F., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropins+and+ovarian+cancer.">Gonadotropins and ovarian cancer.</a> Endocr Rev. 28, 440-461 (2007).</li><li>Fathalla, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incessant+ovulation--a+factor+in+ovarian+neoplasia.">Incessant ovulation--a factor in ovarian neoplasia.</a> Lancet. 2, 163-163 (1971).</li><li>Espey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+the+hypothesis+that+mammalian+ovulation+is+comparable+to+an+inflammatory+reaction.">Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction.</a> Biol Reprod. 50, 233-238 (1994).</li></ol>

There is nothing to disclose.

The development of a three-dimensional organ culture system utilizing alginate hydrogels represents a versatile method for analyzing many different tissue types from a wide variety of organisms. The use of 3D cultures can be extended to the fields of tissue engineering and regenerative medicine as it provides a scaffold on which cells can grow11. Currently, our laboratory is undergoing initial studies using human ovarian and fallopian tube tissue; however, culture of human and non-human primate follicles has b...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;MEM medium</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Novamatrix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Sigma Aldrich</td>
			<td>C0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene mesh fiber</td>
			<td>McMaster Carr</td>
			<td>&nbsp;</td>
			<td>45" wide roll; 0.0041" opening</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alginate lyase</td>
			<td>Sigma Aldrich</td>
			<td>A1603</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1603</td>
			<td>Sigma Aldrich</td>
			<td>C9697</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedicals</td>
			<td>103700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>Gibco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ITS solution</td>
			<td>Roche</td>
			<td>11074547001</td>
			<td>Insulin/transferrin/sodium selenite</td>
		</tr>
		<tr>
			<td>Cytokeratin 8 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-1</td>
			<td>Use at 1:200</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>Sigma</td>
			<td>B50002-1G</td>
			<td>Final concentration 10 &mu;M</td>
		</tr>
	</tbody>
</table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K 조회수.  University of Illinois at Chicago. 그들의 입체적인 맥락에서 정상 세포의 문화 세포 변형 및 tumorigenesis에 필요한 초기 이벤트를 연구하는 다른 방법을 나타냅니다. 이 방법은 난소암 형성 초기에 이벤트를 연구 정상 난소와 oviductal 세포를 성장하는 데 사용됩니다.

비디오: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...