We are grateful to Dr. K. Sekiguchi (Institute for Protein Research, Osaka University), Dr. M. Yamada (Institute for Protein Research, Osaka University), Dr. T. Ikejima (China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University) and T. Hayashi (China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University) for helpful comments.

1. Preparation of keratinocyte culture medium
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Add 5 mL of penicillin-streptomycin and 10 mL of additive supplement K-1 to 500 mL of K110 Type-II medium (K110) using a pipette.</li>
</ol>
2. Preparation of the fibril form of type I collagen
NOTE: Perform procedures until step 2.6 under aseptic conditions.
<ol>
	<li>Keep 10x phosphate-buffered saline (PBS (-)), deionized water, collagen, a 96-well culture plate, and an empty 2-mL tube on ice. 
	NOTE: Do not use the same plate to prepare the fibril and non-fibrous forms.</li>
	<li>Add 1.12 mL of deionized water to an empty 2 mL tube using a pipette. Add 200 &#181;L of 10x PBS (-) to the deionized water in the 2 mL tube using a pipette. Gently shake the tube several times.</li>
	<li>Add 0.66 mL of collagen to the 2 mL tube using a pipette. Gently and quickly shake the tube several times. 
	NOTE: Avoid bubble formation in the solution as much as possible. Prepare collagen solution immediately prior to use.</li>
	<li>Pour 100 &#181;L of the collagen solution from step 2.3 into each well of the 96-well culture plate. Gently shake the culture plate in a left to right motion. 
	NOTE: Cover the entire surface of the wells with the collagen solution. If the solution does not cover the entire surface, spread the solution using a pipette tip. Avoid bubble formation in the solution as much as possible.</li>
	<li>Place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 1 h. Check the gelation of collagen and move the culture plate to a clean bench. 
	NOTE: Confirm gelation by tilting the&#160;culture plate.</li>
	<li>Gently pour 150 &#181;L of K110 on the gels using a pipette along the wall of the well, place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 1 h. Move the culture plate to a clean bench. Prior to cell culture, gently discard the K110 using a pipette. 
	NOTE: To protect the gels, the tip of the pipette should touch the wall of the well.</li>
</ol>
3. Preparation of the non-fibrous form of type I collagen
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Add 4 &#181;L of collagen to 1.2 mL of 1 mM hydrochloric acid (HCl) in a 1.5 mL tube and gently mix using a pipette. 
	NOTE: Prepare collagen immediately prior to use. Keep collagen and HCl chilled until the time of use.</li>
	<li>Pour 100 &#181;L of collagen into each well of a 96-well culture plate using a pipette. Gently shake the culture plate in a left to right motion and incubate at room temperature for 1 h. 
	NOTE: Ensure that the entire surface of the wells is covered with solution.</li>
	<li>After the incubation, discard the collagen solution and wash the wells with PBS (-) twice using a pipette.</li>
	<li>Pour 150 &#181;L of 1% bovine serum albumin/PBS (-) (1% BSA) to a well of the 96-well culture plate and incubate at room temperature for 1 h. Prior to cell culture, discard the 1% BSA. 
	NOTE: Ensure that the entire surface of the wells is covered with solution.</li>
</ol>
4. Culture of FEPE1L-8 cells
NOTE: Perform all procedures under aseptic conditions.
<ol>
	<li>Maintain FEPE1L-8 cells in K110 in a 100 mm culture dish in a CO2 incubator and incubated at 37 &#176;C and 5% CO2, to semi-confluency.</li>
	<li>Prepare K110, trypsin, and trypsin inhibitor in a water bath at 37 &#176;C.</li>
	<li>Carefully remove the medium from the culture dish, add 3 mL of 0.05% trypsin using a pipette, place the dish in a CO2 incubator and incubate at 37 &#176;C for 5 min. After incubation, check cell detachment from the surface of the culture dish using phase-contrast microscopy at 10x magnification. 
	NOTE: The morphology of the detached cells becomes round. If the cells spread, incubate for an additional period of 5 min.</li>
	<li>Add 3 mL of trypsin inhibitor and collect the detached cells in a 15 mL centrifuge tube using a pipette. Centrifuge the cells in a 15 mL centrifuge tube at 200 x g for 5 min.</li>
	<li>Discard the supernatant and re-suspend the pellet in 10 mL of K110 using a pipette. Count the cells using phase-contrast microscopy at 10x magnification and prepare cell concentration at 5.0 x 104&#160;cells/mL by appropriate dilution with K110.</li>
	<li>Gently seed 0.1 mL of K110 with cells in each well of the culture plate using a pipette along the wall of the well. Place the culture plate in a CO2 incubator and incubate at 37 &#176;C for the indicated time (2 h, 1 day, 3 days). 
	NOTE: To protect the gels, the tip of the pipette should touch the wall of the well.</li>
</ol>
5. Estimation of the number of viable cells
<ol>
	<li>Incubate K110 in a water bath at 37 &#176;C. Mix 130 &#181;L of tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H&#160;tetrazolium, monosodium salt (WST-8), and 1.3 mL of K110 in a 2-mL tube using a pipette.</li>
	<li>Move the culture plate to a clean bench and gently discard the K110 using a pipette. Gently wash away non-adherent cells with K110 by using a pipette</li>
	<li>Add 110 &#181;L of K110 mixed with WST-8 in each well using a pipette, place the culture plate in a CO2 incubator and incubate at 37 &#176;C for 2 h.</li>
	<li>Move the culture plate to a clean bench, collect 100 &#181;L of the conditioned medium from each well, and move it into a well of another 96-well culture plate. Measure absorbance at a wavelength of 450 nm (OD450) using a microplate reader and estimate the number of viable cells.</li>
</ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+extracellular+matrix+and+its+components+in+culture.">Use of extracellular matrix and its components in culture.</a> Extracellular Matrix: A Practical Approach, 1st ed. , 289-301 (1995).</li><li>Yamato, M., Hayashi, T., Ninomiya, Y., Olsen, B. 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K., Cone, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+primary+human+epithelial+cells+by+cloned+cervical+carcinoma+DNA+containing+human+papillomavirus+type+16+E6/E7+open+reading+frames.">Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/E7 open reading frames.</a> Journal of General Virology. 70, 1261-1266 (1989).</li><li>Kaur, P., Carter, W. 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D., Brooks, P. C., Weinberg, K. I., DeClerck, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+with+fibrillar+collagen+inhibits+melanoma+cell+proliferation+by+up-regulating+p27KIP1.">Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (18), 10026-10031 (2000).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+levels+of+reactive+oxygen+species+in+murine+preadipocyte+3T3-L1+cells+cultured+on+type+I+collagen+molecule-coated+and+gel-covered+dishes+exert+opposite+effects+on+NF-&#954;B-mediated+proliferation+and+migration.">Differential levels of reactive oxygen species in murine preadipocyte 3T3-L1 cells cultured on type I collagen molecule-coated and gel-covered dishes exert opposite effects on NF-&#954;B-mediated proliferation and migration.</a> Free Radical Research. 28, 1-16 (2018).</li></ol>

The authors declare that they have no competing financial interests.

Some ECM components, including type I collagen, form three-dimensional structures in vivo1. Culturing on such a three-dimensional, gel substrate provides more physiological conditions in vitro than on a two-dimensional, plastic surface1,2,3,4. Numerous protocols regarding the gel culture method have been reported, such as using type I collagen1,2,3,4,6,7,11, type IV collagen14,15, and Matrigel16. Type I collagen is a well-defined and widely used material because of its abundance and ease of handling. The features of purified type I collagen depend on the animal species, age, and purification methods5,17. Type I collagen can be purified using acetic acid and/or proteases, such as pepsin, papain, and proctase5,17. Acid-soluble collagen maintains amino-telopeptides and protease-soluble collagen are cleaved amino-telopeptides5,17. The reserved length of amino-telopeptides depends on the type of proteases, and the presence of telopeptides affects fibril morphology and gel strength5,17. The viscosity of acid-soluble collagen fibrils is greater than that of protease-treated collagens17. In this study, we used acid-soluble bovine type I collagen. Pepsin-solubilized collagen can also be used in this gel culture protocol; however, the gel strength is weaker17. These differences in materials can cause cell behavioral differences, but they are currently not well understood.
The gel culture protocol described in this study is very simple. Many modifications of this method have been reported. One possible modification for the culture of keratinocytes is to mimic the basement membrane. Type IV collagen gels may be better to keep a basement membrane-like substrate structure in vitro15,18. However, a long incubation period is required for the preparation of type IV collagen gels14,15,18. Instead, mixing type IV collagen with type I collagen gels can produce novel culture substrates (type I/type IV collagen hybrid gels)19. These hybrid gels are easy to handle, require a short time for gelation, and yield more basement membrane-like conditions for keratinocytes. On type I/type IV collagen hybrid gels, keratinocytes survive, form colonies, and induce terminal differentiation19. This hybrid method has versatile applications.
On-gel culture using type I collagen affects cancer cells. On the fibril form of type I collagen, Akt activation and growth of Caco-2 cells (a colon cancer cell line) are suppressed7. In addition, the growth of human melanoma cells (M24met) on the fibril form is arrested at the G1/S checkpoint20. Moreover, markedly increased levels of reactive oxygen species are observed in murine 3T3-L1 preadipocytes cultured on the fibril form. Furthermore, cell proliferation and migration are stimulated in opposite directions by the non-fibrous and fibril forms of type I collagen21.

Interstitial connective tissues comprise a three-dimensional protein meshwork of heterogeneous composition, primarily composed of type I collagen fibrils1. Collagen fibrils play a key role as a scaffold for cells1,2,3 and interact with other extracellular matrix (ECM) proteins3. In vitro, different forms of type I collagen can be used as substrates for cell culture depending on the handling method1,2,3,4. Under acidic conditions, type I collagen maintains the non-fibrous form5. Coating the surface of culture dishes with the non-fibrous form promotes cell adhesion and proliferation6,7. At physiological pH and temperature, type I collagen molecules reassemble into fibrils that form gels possessing a three-dimensional structure1,2,3,4,5,6,7,8. There are several important differences between the fibril and non-fibrous forms of type I collagen, including matrix stiffness and efficiencies of reconstruction of ECM components by the cells during the culture1. Matrix stiffness is one of the most studied regulatory factors of cell culture1, 9. However, the complex interactions between substrates and cells remain to be clarified. To examine the complex interactions between cells and environmental factors, a simple system is useful. Comparison of the cellular behavior on the two different forms of collagen may help to simplify the effect of environmental factors. Depending on the purpose of their use, different forms of type I collagen can be selectively used. Normally, keratinocytes are in contact with the basement membrane but not with type I collagen. However, during wound healing, keratinocytes move to the dermal connective tissue, proliferate, and heal the wound10.
Recently, we demonstrated that the concentration of extracellular calcium is important for proliferation of keratinocyte line cells by using the culture system on the fibril form of type I collagen mimicking the dermal connective tissue11. When the keratinocyte cell line FEPE1L-8 was cultured on the fibril form of type I collagen, the shape of the cells was round and their proliferations were stopped at an extracellular calcium concentration of 30 &#956;M11. When the calcium concentration was increased to 1.8 mM, cell growth was recovered11. The cells grew under both calcium concentrations (30 &#956;M and 1.8 mM) when cultured on the non-fibrous form11, whereas they were more sensitive to the exogenous calcium concentration when cultured on the fibril form. FEPE1L-8 was generated through transfection with the papillomavirus type 16 transforming genes E6 and E7 from human cervical carcinoma, non-tumorigenic, inhibit unlimited proliferation with limited differentiation potential like normal keratinocytes12,13. FEPE1L-8 cells can be maintained by using some kinds of keratinocyte specific medium, including K110 Type-II with additive supplement K-1 (K110)6. Here, we describe the culture protocol of the human keratinocyte cell line on the non-fibrous and fibril forms of type I collagen.

<table><tbody><tr><td>Albumin, from bovine serum (BSA)</td><td>Merck KGaA</td><td>A4503</td><td></td></tr><tr><td>1 % BSA&amp;nbsp;</td><td>Merck KGaA</td><td>A4503</td><td>Dissolve 1 g of BSA powder in 100 mL of PBS (-) and filtrate&amp;nbsp;</td></tr><tr><td>Cell Counting Kit-8</td><td>Dojindo Molecular Technologies, Inc.</td><td>347-07621</td><td>tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H&amp;nbsp;tetrazolium, monosodium salt (WST-8)&amp;nbsp;</td></tr><tr><td>Collagen type I (Acid soluble collgen)</td><td>Nippi Inc.</td><td>ASC-1-100-20</td><td>from bovine (any other species available, concentration is 3.0 mg/mL</td></tr><tr><td>disposable membrane filter unit DISMIC cellulose acetate</td><td>ADVANTEC Co., LTD.</td><td>25CS020AS</td><td>0.2 &amp;micro;m pore size</td></tr><tr><td>human keratinocyte line cell&amp;nbsp; FEPE1L-8</td><td></td><td></td><td>&amp;nbsp;Donated Dr.W.G. Carter (Fred Hutchinson Cancer Research Center, Seattle, WA)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>K-1</td><td>Kyokuto Seiyaku Inc.</td><td>28204</td><td>additive supplement with 500 mg of BSA, 500 mg of bovine pituitarybody extracts, 2.5 mg of insulin, 0.05 &amp;micro;g of h-EGF and 5 mg of heparin</td></tr><tr><td>K110 Type-II medium</td><td>Kyokuto Seiyaku Inc.</td><td>28204</td><td>keratinocyte basal culture medium&amp;nbsp;</td></tr><tr><td>K110 Type-II medium with K-1 and Penicillin-Streptomycin (K110)</td><td>Kyokuto Seiyaku Inc.</td><td>28204</td><td>Add 5 mL of Penicillin-Streptomycin and 10 mL of additive supplement (K-1) in 500 mL of K110 type-II keratinocyte basal culture medium&amp;nbsp;</td></tr><tr><td>PBS (-)&amp;nbsp;</td><td>Merck KGaA</td><td>P-5368</td><td>Dissolve 1 pouch of PBS (-) powder in 1000 mL of deionized water and filtrate&amp;nbsp;</td></tr><tr><td>10x PBS (-)</td><td>Merck KGaA</td><td>P-5368</td><td>Dissolve 1 pouch of PBS (-) powder in 100 mL of deionized water and filtrate&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin</td><td>MP Biomedicals, LLC</td><td>1670049</td><td>10000 units/mL of penisillin G and 10,000&amp;mu;g/mL of streptmycin sulfate in physiological saline</td></tr><tr><td>Phosphate bufferred saline BioPerformance CertiCertified, pH 7.4</td><td>Merck KGaA</td><td>P-5368-10 pack</td><td></td></tr><tr><td>Trypsin from porcine pancreas</td><td>Merck KGaA</td><td>T4799</td><td></td></tr><tr><td>0.05 % trypsin&amp;nbsp;</td><td>Merck KGaA</td><td>T4799</td><td>Dissolve 0.25 g of&amp;nbsp; trypsin and&amp;nbsp; 0.186 g of EDTA.2Na in 500 mL of PBS (-)&amp;nbsp; and filtrate&amp;nbsp;</td></tr><tr><td>Trypsin inhibitor from soybean</td><td>FUJIFILM Wako Pure Chemical corporation</td><td>202-20123</td><td></td></tr><tr><td>Trypsin inhibitor&amp;nbsp;</td><td>FUJIFILM Wako Pure Chemical corporation</td><td>202-20123</td><td>Dissolve 50 mg of trypsin inhibitor and 500 mg of BSA in 500 mL of PBS (-) and filtrate&amp;nbsp;</td></tr></tbody></table>

evaluation of keratinocyte proliferation on two- and three-dimensional type i collagen substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

A schematic representation of the treatment of the surfaces of culture dishes using type I collagen is depicted in Figure 1. Cell morphologies observed on the non-fibrous and fibril forms are presented in the left- and right-side panels of Figure 2, respectively. FEPE1L-8 cells were cultured for 2 h (upper panels) and 3 days (lower panels). In the initial 2 h of culturing, the cells adhered and spread on both forms of collagen (Figure 2, upper panels). Three days after seeding, cells on the non-fibrous form continued to spread and cell numbers increased (Figure 2, lower left panel). In contrast, the cells on the fibril form showed limited spreading (Figure 2, lower right panel). FEPE1L-8 cells continued to proliferate on the non-fibrous form of type I collagen (Figure 3, solid black line, closed black circles) and on the untreated dish surfaces (Figure 3, dotted gray line, closed gray circles). In contrast, cells did not proliferate on the fibril form (Figure 3, dotted line, open circles). Figure 2 and Figure 3 have been modified from Fujisaki et al11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59339/59339fig1v2.jpg" /> 
Figure 1: Schematic representations of culture dish surfaces treated with type I collagen. Under acidic conditions, type I collagen molecules are adsorbed on the surface of a dish in the non-fibrous form (left panel). Under neutral conditions at 37 &#176;C, type I collagen molecules are reassembled into fibrils and adsorbed on the surface of dishes in the gel form (right panel). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59339/59339fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59339/59339fig2.jpg" /> 
Figure 2: Morphology of FEPE1L-8 cells. FEPE1L-8 cells in K110 were cultured using the non-fibrous form (10 &#181;g/mL; left panels) or fibril form (1 mg/mL; right panels) of type I collagen for 2 h (upper panels) or 3 days (lower panels). White bars indicate 100 &#181;m. Figure 2 has been modified from Fujisaki et al. in Figure 1E&#8211;H11. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59339/59339fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59339/59339fig3v3.jpg" /> 
Figure 3: Proliferation of FEPE1L-8 cell. The number of viable cells were estimated on the non-fibrous form (black solid line, black filled circles) or on the fibril form (dotted line, open circle) of type I collagen, or untreated dish surfaces (gray dotted line, gray filled circles) for 2 h, 1 day, and 3 days. Experiments were performed in triplicates and values are shown as means + SD. This figure has been modified from Fujisaki et al. in Figure 1J11. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59339/59339fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates at JoVE.com

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

evaluation-keratinocyte-proliferation-on-two-three-dimensional-type-i

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.0K Views.  Nippi Research Institute of Biomatrix. Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Video: Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Cancer Research

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 In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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 ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Developmental Biology

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies

Source: Morris, R. J. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58701"> Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture</a>. J. Vis. Exp. (2019)
In this video, we perform a clonogenic assay of keratinocytes harvested from adult mice to determine the ability of individual cells to proliferate and form colonies. This keratinocyte harvesting technique is useful for studying other downstream aspects of cutaneous chemical carcinogenesis and tumor promotion.

Source: Morris, R. J. et al.  Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture. J. Vis. Exp. (2019)
In this video, we ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Summary

Explore More Videos

Chapters in this video

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

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

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

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

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies