Name: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells (Video) | JoVE Journal
Uploaded: 2010-12-13T17:00:00.000+00:00
Duration: 9 min 28 s
Description: Learn to fabricate modular micro-tissues using collagen gel modules embedded with cells for tissue engineering applications. This method enables in vitro cellular assays and in vivo implantation studies.

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5 minutes

Funding was provided by the National Institutes of Health (EB 001013), Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research.  We thank Dr. A. P. McGuigan for her expertise and help in development of modules.

1) Preparation of Tubing
<ol>
<li>	Cut 2 to 3 m lengths of polyethylene tubing (0.76 mm I.D. x 1.22 mm O.D.).</li>
<li>	Thread a 20 G1 needle into one end of the tubing.</li>
<li>	Coil the tubing and place in a convertors self-seal pouch (7 1/2" x 13"). The two ends of the tubing should be placed at the unsealed end of the bag and taped in place with gas sterilization tape.</li>
<li>	Seal bag and gas sterilize.</li>
</ol>
2) Neutralization of Collagen
Note: 1 ml of collagen fills about 2 m of polyethylene tubing.
<ol>
<li> Place the necessary amount of collagen needed for the desired length of tubing into a 15 ml conical tube and keep on ice. </li>
<li> Add 128 uL of 10x α-MEM per mL of collagen to the tube and mix by repeatedly pipetting the solution. Note: (1) The solution should turn yellow as the α-MEM is mixed into the acidified collagen and (2) try to minimize bubbles when mixing.</li>
<li> Neutralize the collagen by adding 0.8 M sodium bicarbonate until the collagen has a pH of 7.4. For a general estimate of the pH add sodium bicarbonate until the yellow collagen solution turns a light pink color. Note: It typically takes 30 to 60 μL per each mL of collagen.</li>
<li> Keep neutralized collagen on ice until ready to use as the neutralized collagen will gel if not kept at 4°C.</li>
</ol>
3) Embedding of Cells (Optional)
Note: Depending on the cell type the cells can be embedded at a concentration of between 1 million cells per mL to 20 million cells per mL. Use the medium required for the cells that you want to embed.
<ol>
<li> Remove media from cells and wash with 5 mL PBS.</li>
<li> Remove PBS and add 3 mL trypsin-EDTA.</li>
<li> Incubate the cells in trypsin-EDTA for 5 min at 37°C.</li>
<li> Resuspend cells in 7 mL media.</li>
<li> Count cells.</li>
<li> Transfer the required number of cells for embedding to a 15 mL conical tube.</li>
<li> Pellet the cells at 300 x g for 5 min.</li>
<li> Aspirate media off the cell pellet.</li>
<li> Resuspend the cells in the neutralized collagen and keep on ice.</li>
</ol>
4) Gelling of Collagen
<ol>
<li> In a biological safety cabinet cut off the sealed end of the bag containing the polyethylene tubing.</li>
<li> Attach a 3 ml syringe to the 20G needle in the one end of the tubing using sterilized tweezers. Keep the tubing in the bag.</li>
<li> Insert the other end of the tubing into the neutralized collagen until the tip is at the bottom of the conical tube using sterilized tweezers. Keep the conical tube on ice.</li>
<li> Pull the collagen into the polyethylene tubing by retracting the plunger of the syringe slowly. 
Note: If the collagen is pulled into the tubing to quickly then bubbles will form in the collagen.</li>
<li> After pulling the collagen through the tubing, pull the 20G needle out of tubing and put both ends back into the bag. Tape the bag shut.</li>
<li> Incubate the bag at 37°C for 60 min. If the collagen contains cells then flip the bag over every 15 min. so that the cells do not settle to one side of the module.</li>
</ol>
5) Cutting of Tubing Containing the Gelled Collagen
<ol>
<li> Autoclave headers and blade for the cutting device and tray to hold tubing.</li>
<li> Assemble the cutting device.</li>
<li> Place tubing in a sterile tray in front of cutting device and load the tubing into the 
cutting device.</li>
<li> Set up a 50 mL conical tube containing 25 mL of media at the outlet of the cutting 
device to collect the cut modules. 
Note: Use media containing FBS even for collagen-only modules as the modules do not separate from the tubing without FBS in the media. If the modules contain cells use the media required for the embedded cells. If there are no embedded cells in the modules use the media for the endothelial cells.</li>
<li> Set the cutting device to cut 2 mm lengths of tubing and cut the tubing.</li>
<li> Incubate cut modules at 37°C for 60 min in the 50 mL conical tube.</li>
</ol>
6) Removing Collagen Modules from Tubing
<ol>
<li> Vortex 50 mL conical tube containing modules in the tubing for 10 sec. 
	Note: Be gentle when separating modules embedded with high cell density to avoid breakage. </li>
<li> Let modules settle to the bottom of the tube. Takes about 5 min.</li>
<li> Using a wide mouth 10 mL serological pipette transfer the settled modules to a 15 mL 
conical tube.</li>
<li> Repeat steps 6.1 to 6.3 twice.</li>
<li> Go to step 7 to coat modules with endothelial cells or transfer modules to a non-tissue 
culture treated plate.</li>
</ol>
7) Coating the modules with endothelial cells
<ol>
<li> Settle modules into the bottom of a 15 mL conical tube with 5 mL of media for the embedded 
cells.</li>
<li> Remove media from the endothelial cells and wash with 5 mL PBS.</li>
<li> Remove PBS and add 3 mL trypsin-EDTA.</li>
<li> Incubate the cells in trypsin-EDTA for 5 min at 37°C.</li>
<li> Resuspend cells in 7 mL media.</li>
<li> Count cells. Need 5x106 endothelial cells per mL of packed modules at the bottom of 
the conical tube.</li>
<li> Pellet the cells at 300 x g for 5 min and resuspend in 5 mL endothelial cell media.</li>
<li> Add the 5 mL of the media containing endothelial cells to the modules. 
Note: This gives a 50:50 ratio of the two media types which we have found to work for the survival and function of both cell types. Test the use a co-culture medium formulated to ensure proper maintenance of phenotypes of the two cells types before using it. </li>
<li> Incubate the modules with the endothelial cells both statically and dynamically.</li>
<li> For static incubation mix modules with endothelial cells by inversion and set the tube upright at 37°C for 15 min. Repeat.</li>
<li> Dynamically seed the endothelial cells by gently rocking the tube at 37°C for 60 min.</li>
<li> Repeat static incubation twice.</li>
<li> Place the modules in a non-tissue culture treated plate and incubate overnight at 37°C.</li>
<li> The next day place the modules in a new non-tissue culture treated plate with fresh media (50:50 mix if using co-culture system) to remove unattached endothelial cells.</li>
<li> Incubate modules that are coated with endothelial cells until they cover 100% of the surface of the modules. This usually takes about 7 days.</li>
</ol>
8) Representative Results:
The modules will look cylindrical when they first are removed from the tube. If the modules contain embedded cells and/or are coated with endothelial cells they can contract up to 50% in volume and develop an oval shape (Figure 1). The modules will also become denser and more opaque when viewed by light microscopy (Figure 1). Also when the endothelial cells are confluent on the surface of the modules there is a formation of tight junctions which can be seen by immunofluorescence staining of VE-Cadherin (Figure 2). Mass transfer analysis has demonstrated that modules are capable of supporting high cell densities (8x107 cells/cm3) without developing a necrotic core due to inadequate oxygen transport, which is often problematic in larger tissues (e.g. modules of large diameter, D = 1.4mm) (Figure 3).
<img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Microscopy: biofilm module growth comparison, Day 0 vs Day 3, size metrics, fluorescent markers." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1: Module fabrication and contraction. Module contraction occurred during the three days following HUVEC-C (endothelial cell line) seeding results significantly smaller module diameter and length (p &lt; 0.001) (A). Embedded HepG2 cells were uniformly distributed within modules at time of fabrication and retained high viability (B). [Live cells green; dead cells red] [adapted from Corstorphine 2010]

<img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Fluorescence microscopy image; cell structure visualization; green fluorescent protein labeling." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2: VE-Cadherin immunofluorescent staining of EC tight cell junctions 10 days after RAEC where coated on the surface of a module. Scale bar = 50 μm.

<img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Cell culture growth comparison, microscopy images showing small and large modules over days." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3: Masson's trichrome staining of typical modules (0.76 mm initial diameter) and large modules (1.4 mm initial diameter) demonstrate the effect of oxygen diffusion limitations. Seven days following fabrication, a large number of dead cells had formed within the core of the large modules (lower right panel), leaving only a thin rim (~200μm thick) of viable cells. Conversely, the small modules retained a uniform and high distribution of live cells. [Modules embedded with HepG2 cells and coated with endothelial cells.] [Taken from Corstorphine 2010]

<img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Microscopy images of tissue; A: fluorescence, B: dark-field; cellular structure analysis." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4: (A) HMEC-1 seeded on poloxamine - collagen modules. After Day 1 of seeding the poloxamine-collagen modules retain their cylindrical shape and there is limited cell attachment properties compared to collagen only modules. Scale bar = 200 μm. (B) Light microscope image of a collagen module containing PLGA-based biodegradable microspheres. Scale bar = 500 μm.

<img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Microbial growth morphology and fluorescence microscopy in biofilm formation analysis." data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5: Examples of in vitro assays. (A) Angiogenesis assay: capillary-like formations on the module seeded with endothelial cells can be easily detected and quantified after 5 days of incubation with adipose-derived stem cells. (B) Confocal microscopy images of a live/dead assay on modules where the staining for live cells is green and for dead cells is red.

<img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Histology microscope image; tissue sample analysis; cellular structure examination; pathology study." data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Figure 6: Collagen modules containing embedded mesenchymal stem cells and a surface layer of endothelial cells. Modules were exposed to shear stress (~0.64 dyn/cm2) for 7 days in a microfluidic chamber and begin to show fusion at points of contact. Endothelial cells are stained with vWF (brown) and mesenchymal stem cell nuclei appear blue (H&amp;E). Scale bar = 100μm.

<img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Microscopic image of tissue showing abnormal growth indicating potential carcinoma." data-altupdatedat="1788235205000" data-alt="Figure 7"> 
Figure 7: Hundreds of collagen modules containing Adipose-Derived Stem cells (ASC) and coated with Human Microvascular Endothelial Cells (HMEC) are implanted under mouse skin to study ASC/HMEC interaction in vivo for fat regeneration applications.

<ol><li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li>
<li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells%5BTitle%5D)+AND+%22J+Tissue+Eng+Regen+Med%22%5BJournal%5D)">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li>
<li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li>
<li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells%5BTitle%5D)+AND+%22Ann+Biomed+Eng%22%5BJournal%5D)">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li>
<li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+%5Bcorrected%5D%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li>
<li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li>
<li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modular+tissue+engineering%3A+fabrication+of+a+gelatin-based+construct%5BTitle%5D)+AND+%22J+Tissue+Eng+Regen+Med%22%5BJournal%5D)">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li>
<li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li>
<li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+criteria+for+a+modular+tissue-engineered+construct%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li>
<li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li>
<li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collagen%2Fpoloxamine+hydrogels%3A+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li>
</ol>

No conflicts of interest declared.

We have fabricated several different micro-tissues using modules embedded with different cell types1-11. We have successfully embedded primary cardiomyocytes, islets, adipose stem cells and mesenchymal stromal cells as well as several cell lines including HepG2, NIH 3T3 and clone 9 liver cells. We have coated the modules with several types of endothelial cells including rat aortic endothelial cells, human umbilical vein endothelial cells and human microvascular endothelial cells. Modules have also been prod...

<table><tbody><tr><td>Intramedic tubing PE60</td><td>BD Biosciences</td><td>427416</td><td>Different diameter of tubing can be used to change the diameter of the modules</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>GIBCO, by Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>GIBCO, by Life Technologies</td><td>25200-072</td><td></td></tr><tr><td>Purcol acidificed collagen, 3 mg/mL</td><td>Cedarlane Labs</td><td>5005-B</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>20G needle</td><td>BD Biosciences</td><td>305175</td><td>Diameter of the needle needs to be similar to the diameter of the tubing</td></tr><tr><td>3 mL syringe</td><td>BD Biosciences</td><td>309585</td><td></td></tr><tr><td>15 mL tube</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>50 mL tube</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>Convertors Self-Seal Pouch 7 1/2&amp;#8221; x 13&amp;#8221;</td><td>Cardinal Health</td><td>92713</td><td></td></tr><tr><td>10 ml Wide Tip serological pipet</td><td>BD Biosciences</td><td>357504</td><td></td></tr><tr><td>10 ml serological pipet</td><td>BD Biosciences</td><td>357551</td><td></td></tr><tr><td>5 ml serological pipet</td><td>BD Biosciences</td><td>357543</td><td></td></tr></tbody></table>

fabrication of micro-tissues using modules of collagen gel containing cells

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

The overall goal of this procedure is to produce modular micro tissues that can be used for tissue engineering experiments. This is accomplished by first pulling the collagen solution into a polyethylene tubing, using a syringe, and then gelling the collagen. The second step is to cut the polyethylene tubing into two millimeter lengths to form the micro tissue modules.The third step of the procedure is to separate the modules from the tubing, and lastly, the modules are coated with endothelial cells. Ultimately, the resulting micro tissues can be used for in vitro cellular assays to study the interaction of cells via a microfluidic chamber. In addition, the micro tissues can be implanted to generate tissues for studying the interaction and remodeling of the micro tissues in vivo.Hi, my name is Brendan Long from the Sepan Lab here at the University of Toronto. A major obstacles in creating large 3D tissue substitute is a lack of rapid vascularization after implantation. In this video, we will show you how to create modular tissue coated with endothelial cells to help overcome this limitation.Demonstrating the procedure will be deemed Chamberlain, a post-op fellow from the Septin lab. The amount of collagen to neutralize will depend on the length of the polyethylene tubing. In this demonstration, we'll use a two meter polyethylene tubing with an inner diameter of 0.76 millimeter and outer diameter of 1.22 millimeter.To prepare the tubing, thread a 20 G one needle into one end of the tubing, and then place the tubing in a converter self-heal pouch for gas sterilization. Since one milliliter of collagen fills about two meters of polyethylene tubing, we will neutralize one milliliter of collagen. Place the collagen into a 15 milliliter conical tube and keep the tube on ice.Add 128 microliters of 10 x alpha, MEM per milliliter of collagen to the tube and mixed by repeatedly pipetting the solution. Taking care to minimize making bubbles, the solution should turn yellow as the alpha MEM is mixed into the acidified collagen. Next, neutralize the collagen by adding 0.8 molar sodium bicarbonate For a general estimate of the pH, add sodium bicarbonate until the yellow collagen solution turns a light pink color.It typically takes 30 to 60 microliters per milliliter of collagen to achieve a pH of 7.4. To prevent the neutralized collagen from gelling, keep it on ice until it is ready to be used. To begin the procedure for jelling the collagen, take the bag containing the sterilized polyethylene tubing to a biological safety cabinet and cut off the sealed end of the bag while keeping the tubing in the bag.Use sterilized tweezers to attach a three milliliter syringe to the 20 gauge needle that was previously inserted into one end of the tubing. Insert the other end of the tubing into the neutralized collagen until the tip is at the bottom of the conical tube, which is kept on ice. This next step you have to do slowly, otherwise, you introduce air bubbles into the collagen.Retract the plunger of the syringe slowly to pull the collagen into the polyethylene tubing. When the collagen has been pulled through the tubing, pull the 20 gauge needle out and put both ends of the tubing back into the bag. After taping the bag shut, incubated at 37 degrees Celsius for 60 minutes.A preassembled cutting device will be used to cut the tubing. To begin this procedure, place tubing in a sterile tray in front of the cutting device and load the tubing into the cutting device at the outlet of the cutting device, set up a 50 milliliter conical tube containing 25 milliliters of endothelial cell media. To collect the cut modules, set the cutting device to cut two millimeter lengths of tubing and then cut the tubing.Incubate the cut modules in the 50 milliliter conical tube at 37 degrees Celsius for 60 minutes. To remove collagen modules from the tubing, vortex, the 50 liter conical tube containing the modules at medium speed. For 10 seconds, let the module settle to the bottom of the tube, which takes about five minutes.A loose pellet of modules will form at the bottom of the tube while the tubing will form a layer on top of the media. After the modules have settled, use a wide mouth, 10 milliliter serological pipette to transfer them to a 15 milliliter conical tube. Vortex, the 15 milliliter conical tube, and then allow the modules to settle for two more times.The settled modules from the second and third vortex are also transferred to the 15 milliliter tube. After all the modules have been removed from the tubing and pooled, bring the volume of media in the tube to five milliliters. The modules are now ready for coating with endothelial cells, which will be shown next to prepare endothelial cells for coating the modules.First, remove the media from the cells and wash with PBS. After removing the PBS, add three milliliters of trypsin EDTA and incubate for five minutes at 37 degrees Celsius. Next, resus, suspend the cells in seven milliliters of media and transfer to a 15 milliliter tube.Count the cells using a hemo cytometer, you will need five times 10 to the sixth endothelial cells per milliliter of packed modules at the bottom of the conical tube. Pellet the cells at 300 G for five minutes. Resuspend the cell pellet in five milliliters of endothelial cell media.Now add five milliliters of media containing endothelial cells to the modules. The modules are incubated with the endothelial cells, both statically and dynamically for static incubation, mixed modules with endothelial cells by inversion, and then set the tube upright at 37 degrees Celsius for 15 minutes. After 15 minutes, invert the tube again and incubated 37 degrees Celsius for another 15 minutes.To incubate the modules with the endothelial cells dynamically, gently rock the 15 milliliter tube at 37 degrees Celsius for 60 minutes. Once incubation with the endothelial cells is complete, transfer the modules to a non tissue culture treated plate and incubate overnight at 37 degrees Celsius on the following day, place the modules in a new non tissue culture treated plate with fresh media to remove unattached endothelial cells. Incubate modules that are coated with endothelial cells at 37 degrees Celsius until the cells cover 100%of the surface of the modules.This usually takes about seven days. The modules will look cylindrical when they're first removed from the tube. If they're coated with endothelial cells, they can contract up to 50%in volume and develop an oval shape, as well as become denser and more opaque when viewed by light microscopy.In these images, the diameter and length of the modules are indicated by d and l respectively. This next image shows embedded Hep G two cells uniformly distributed within modules at the time of fabrication. The cells retained high viability as indicated by the green color of live cells.Dead cells would be red in color when the endothelial cells are confluent. On the surface of the modules, there's a formation of tight junctions, which can be seen by immunofluorescence. Staining of VE cadherin mass transfer analysis has demonstrated that modules are capable of supporting high cell densities without developing a necrotic core due to inadequate oxygen transport, which is often problematic in larger tissues.In this example, massen Tri chro staining of typical small modules show that the modules retained a uniform and high distribution of live cells seven days following fabrication. In contrast, a large number of dead cells had formed within the core of the large modules leaving only a thin rim of viable cells. Modules have also been produced with a mixture of collagen and a ine polymer or containing drug eluting microspheres.Here when h hm EC one cells are seated on palin collagen modules, the modules retain their cylindrical shape and there is limited cell attachment properties compared to collagen only modules. A light microscope image of a collagen module containing PLGA based biodegradable microspheres is shown here. Several in vitro assays have been shown to be compatible with modules.For example, in an angiogenesis assay, capillary like formations on a module seated with endothelial cells can be easily detected and quantified after five days of incubation with adipose derived stem cells. In another example, confocal microscopy images of a live dead assay on modules shows live cells stained green and dead cells stained red. Finally, modules have also been used in microfluidic chambers and implanted into mice and rats to study the interaction between different cell types and how the micro tissues are remodeled by the host response.Following the procedure, the modules can be cultured in a Petri dish or other vessels such as a bioreactor or a microfluidic device. This method can also be adapted to create different tissue by using various combinations of cell type or using tubings with different diameter. Thanks for watching.

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts.  These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabrication of Myogenic Engineered Tissue Constructs

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.

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

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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.

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

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 µm in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Learn to fabricate modular micro-tissues using collagen gel modules embedded with cells for tissue engineering applications. This method enables in vitro cellular assays and in vivo implantation studies.

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

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