1. Establishment of fibroblast cultures from skin explants
<ol>
	<li>4 mm punch biopsies acquired from human forearm are placed in MEM supplemented 100 units / ml penicillin, 100 &mu;g / ml streptomycin, and 0.25 &mu;g / ml Fungizone.</li>
	<li>Trim any subcutaneous fat and finely chop the biopsy into small pieces by rocking a number 24 scalpel blade against the bottom of the petri dish.</li>
	<li>Place tissue slurry in a 25 cm2 tissue culture flask with primary fibroblast growth media (MEM supplemented with 10% FCS, 100 units / ml penicillin, 100 &mu;g / ml streptomycin, and 25 &mu;g / ml Fungizone) added until the surface of flask is covered but liquid depth is insufficient for tissue to float.</li>
	<li>Following 3 days incubation at 37&deg;C in a humidified atmosphere of 5% CO2 , add 3 ml of primary fibroblast growth media.</li>
	<li>Following another 3 days incubation change media with fresh primary fibroblast growth media, or if cells are nearly confluent they may be split 1:4 into fresh media. (Fungizone can be omitted from this point onwards).</li>
</ol>
2. Stage I - Preparation of collagen I from rat tails
Note: Protocol for approximately 12-14 adolescent (fresh or frozen) rat tails
<ol>
	<li>Prepare rat tail by washing in 70% ethanol and remove tendons as follows:</li>
	<li>Remove skin from rat tail by slicing in the middle of the tail from top to bottom with a scalpel and pealing along the length of the tail.</li>
	<li>Detach tendon from the core of the proximal region of the tail.</li>
	<li>Remove tendon toward the distal region of the tail avoiding the sheath using toothed forceps.</li>
	<li>Extract 1 g of tendon / 250 ml of 0.5 M acetic acid by stirring at 4&deg;C for 48 h.</li>
	<li>Centrifuge the extract (7,500 x g) for 30 mins and discard the pellet.</li>
	<li>Add an equal volume of 10% (w/v) NaCl to the supernatant, and stir for 30-60 min.</li>
	<li>Centrifuge (10,000 x g) for 30 mins, discard the supernatant, and re-dissolve the precipitate in 0.25 M acetic acid at &tilde; 1:1 ratio by stirring for 24 h at 4&deg;C.</li>
	<li>Dialyze the collagen solution against 6-8 changes of 6L &tilde;17.5 mM acetic acid (1 ml glacial acetic acid per liter of cold water, change 2 x daily).</li>
	<li>Centrifuge dialysed collagen at 30,000 x g for 1.5 hrs.</li>
	<li>Remove supernatant and place in sterile flask.</li>
	<li>Adjust the collagen I concentration to &tilde;2 mg/ml using 0.5 mM acetic acid at 4&deg;C.</li>
</ol>
3. Stage II - Setting up the 3D matrix with embedded fibroblasts (allow to contract for 8 days).
<ol>
	<li>Assemble the mix using cold reagents at 4&deg;C in a pre-chilled bottle. Keep collagen I on ice.</li>
	<li>For one T75 flask of confluent primary fibroblasts (&tilde; cell number 1 x 106) use:
	<ul>
		<li>25 ml rat tail collagen (approx conc 2mg/ml)</li>
		<li>3 ml 10xMEM</li>
		<li>0.22 M NaOH: first, add 2ml, then drop-wise whilst stirring, until the collagen turns orange,</li>
		<li>but not pink (usually up to 3 ml). Approximate pH = 7.2.</li>
		<li>Note: It is important to ensure that the medium/gel remains neutral or slightly acidic as fibroblasts will not contract the gel if exposed to even slightly alkaline conditions.</li>
	</ul>
	</li>
	<li>Trypsinise the fibroblasts, spin at 400 x g for 5 mins and remove supernatant.</li>
	<li>During 5 mins spin above: Prepare 12 x 35 mm plastic dishes - normally achieve 12 dishes from one flask of fibroblasts/collagen mix.</li>
	<li>Re-suspend fibroblasts in 3 ml FCS and immediately add to the collagen mix and stir.</li>
	<li>Plate approximately 2.5 ml of collagen/fibroblast per dish as quickly as possible. Try to avoid bubbles and collagen setting.</li>
	<li>Let the collagen set at 37&deg;C in a humidified atmosphere of 5% CO2 in air for 10 mins.</li>
	<li>Add 1ml of fibroblast growth media (DMEM+ 10% FCS).</li>
	<li>Detach the collagen/fibroblast matrix from the sides of the dish using a pipette.</li>
	<li>Next day: Add 1ml of fibroblast growth media (DMEM + 10% FCS).</li>
	<li>Change media every other day. Allow collagen/fibroblast matrix to contract for approximately 8 days, until they fit in a 24-well dish (Contraction from &tilde;3.5 cm to 1.5 cm in diameter, see Figure 1).</li>
</ol>
Note: The collagen concentration should be adjusted to suit the application. The more dilute the collagen solution, the faster it will be contracted by the fibroblasts. Slight differences in contraction rate will be experienced with different batches of collagen at the same concentration. Similarly, different fibroblast cultures will contract the collagen gels at different rates, and the more fibroblasts present, the faster the rate of contraction. Collagen concentration and fibroblast density can therefore by adjusted to modify the rate of contraction, and the final density of the collagen gel.
4. Stage II - Plating cells of interest on top of the matrix
Note: Sterilize all forceps and equipment with ethanol before use.
<ol>
	<li>Using blunt forceps, gently move contracted matrix to 24-well dish. Make sure it does not fold.</li>
	<li>Prepare suspension of cells of interest at approximately 4 x 104 /ml and plate 1ml (after trypsinising, spin cells to get rid of trypsin) on top of the matrix. Actual number of cells needed will vary depending on cell type used. Cell medium should be normal growth medium for the cells of interest.</li>
	<li>Allow cells to grow to confluence on top of the matrix, approximately 3-5 days.</li>
</ol>
5. Stage III - Transferring the matrix to a grid for invasion (approximately 0-21 days)
<ol>
	<li>Cut stainless steel grids to create a tripod and autoclave prior to use (See Figure 2).</li>
	<li>Place sterile grid in 6 cm dish, add growth media to a level above the grid (approx 10.5ml). Place matrix on the grid and gently aspirate media so the bottom of the matrix is in contact with media but not submerged. This is referred to as the air/liquid interface, which creates a gradient that promotes invasion. Replace medium every two days (See Figure 2).</li>
	<li>Live cell, contextual imaging using fluorescent cells can be imaged at this stage or earlier. The contracted or fibrillar collagen I can be imaged using second harmonic generation (SHG, See figure 3). Using multi-photon excitation combined with wide-field detection we find that SHG can be imaged at least 100 &mu;m into the matrix, whereas cells expressing cytoplasmic GFP can be imaged at least 200 &mu;m deep.</li>
</ol>
Note: With respect to quantification of invasion, the day on which matricies are placed on the grids defines Day 0. Placement on grids generates a gradient of cell culture media that promotes invasion into the matrix. Samples can be imaged over the next 1 - 21 days (or longer) to assess biological processes such as invasion, proliferation, survival or differentiation (See reference list).
6. Stage IV - Fixation
<ol>
	<li>Add 5 ml of 4% PFA in a Falcon tube.</li>
	<li>Transfer the matrix onto a flat surface. Cut the matrix in half with a fresh clean scalpel (as you will be staining the cross section), lift the matrix with scalpel and transfer to 4% PFA, fix over night.</li>
	<li>Organotypic matrices are now ready to stain with antibody/stain of choice (See Figure 3).</li>
</ol>
7. Representative results:
<img alt="Figure 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> 
Figure 1 Example of fibroblast-contracted collagen I. Mixture of fibroblast and rat tail collagen detached from the petri dish and allowed to contract over 6-8 days.
<img alt="Figure 2" src="/files/ftp_upload/3089/3089fig2.jpg" /> 
Figure 2 Organotypic assay progression. A, schematic of organotypic set up and progression. B, Example of collagen/fibroblast matrix on top of stainless steel, sterile grid to create air/liquid interface.
<img alt="Figure 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> 
Figure 3 Applications of organotypic assay. A, B non-invasive and invasive pancreatic ductal adenocarcinoma (PDAC) cells invading over time with organotypic matrix. C, living skin equivalent showing a stratified epidermis on a fibroblast-contracted collagenous dermal component. D, C8161 melanoma cells invading in to a fibroblast-contracted collagenous dermal equivalent. E, invasive PDAC cells (green) interacting and invading with fibroblasts (red) within the organotypic matrix. F, invasive PDAC cells (green) interacting with a degrading surrounding extracellular matrix (purple) visualised using multiphoton -based second harmonic generation.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

We have nothing to disclose.

Here we present a method for production of a 3-dimensional matrix suitable for studies of invasive cell migration 6-8. The matrix consists of collagen type 1 fibrils, which are contracted over a period of several days by primary human epidermal fibroblasts. The collagen is prepared by acid extraction, not enzymatic digestion, which preserves reactivity at the poly-peptide ends and promotes cross-linking of collagen fibrils into larger aggregates upon neutralization of buffer conditions 9. This results in a re-constituted gel which more closely mimics the features of collagen in vivo and has important consequences for the invasive properties of cells cultured in the gels. It does not matter whether fresh or frozen starting material is used, but use of adolescent rat tails is important because collagen cross-linking is more labile in younger animals. Collagen I from younger animals is therefore easier to extract, and re-constitutes with higher fidelity.
The collagen matrix can be fixed and stained with antibodies directed against either invasive tumor cells or stromal fibroblasts 2,10 and are highly useful for testing the efficacy of drugs which might inhibit invasion 5,6.
Although this protocol suggests the use of primary human skin fibroblasts, it is feasible to use primary fibroblasts from other tissue types, according to the type of tissue under investigation. It is also possible to establish cultures using immortalized fibroblasts, including cells which have been stably transfected with fluorescent protein conjugates. In this way both stromal and tumor cells can be labelled to visualise cell-cell interactions during invasion 11.

<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>10x MEM</td>
<td>Gibco</td>
<td>21430</td>
<td>-</td>
</tr>
<tr>
<td>NaOH</td>
<td>Sigma</td>
<td>367176-500G</td>
<td>Prepare 0.22 M stock in water</td>
</tr>
<tr>
<td>FCS</td>
<td>PAA Laboratories</td>
<td>A15-101</td>
<td>-</td>
</tr>
<tr>
<td>35 mm dishes</td>
<td>Falcon</td>
<td>353001</td>
<td>For step 3.4</td>
</tr>
<tr>
<td>60 mm dishes</td>
<td>Falcon</td>
<td>353004</td>
<td>For step 5.2</td>
</tr>
<tr>
<td>Spring forceps blunt</td>
<td>Samco</td>
<td>E003/02</td>
<td>Toothed, not smooth</td>
</tr>
<tr>
<td>16% paraformaldehyde</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in PBS prior to use</td>
</tr>
<tr>
<td>Screens for cd-1, size 40 mesh</td>
<td>Sigma</td>
<td>S07707-5EA</td>
<td>Stainless steel grids, 5 per pack</td>
</tr>
<tr>
<td>Dialysis tubing</td>
<td>Medicell International</td>
<td>7607 2295</td>
<td>12 - 14 kD</td>
</tr>
<tr>
<td>PBS</td>
<td>Oxoid</td>
<td>BR0014G</td>
<td>-</td>
</tr>
<tr>
<td>Acetic Acid</td>
<td>Sigma</td>
<td>242853</td>
<td>-</td>
</tr>
<tr>
<td>24 well dish</td>
<td>Falcon</td>
<td>353047</td>
<td>For step 4.1</td>
</tr>
<tr>
<td>Fungizone</td>
<td>In vitrogen</td>
<td>15290018</td>
<td>-</td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

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

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 ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

Research

JoVE Journal

Bioengineering

22.4K 조회수.  University of Glasgow. 방법은 콜라겐 유형 I 및 기본 인간 섬유 아세포로 구성된 3 차원 매트릭스의 준비를 위해 설명되어 있습니다. 이 조직 기질의 기본 기능을 모방하고 현미경의 여러 형태의 의무이기 때문 organotypic 젤은 침입 세포 마이그레이션을 평가하는 유용한 기판 역할을합니다.

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

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.