The authors wish to thank the personnel of the Laboratory Animal Facility of the Experimental Medicine Unit, Nurse Carolina Ba&#241;os G. for technical and surgical support, Marco E. Gudi&#241;o Z. for support in microphotographs, and Erick Apo for support in liver histology. The National Council supported this research for Science and Technology (CONACyT), grant number SALUD-2016-272579 and the PAPIIT-UNAM TA200515.

The present research was approved by the ethics committee of the School of Medicine (DI/115/2015) at the Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM) and the ethics committee of the Hospital General de Mexico (CI/314/15). The institution fulfills all technical specifications for the production, care, and use of laboratory animals and is legally certified by national law (NOM-062-ZOO-1999). Male Wistar rats weighing 150-250 g (6-8 weeks old) were obtained from the Laboratory Animal Facility of the School of Medicine, UNAM, for this study.
1. Obtaining collagen matrix scaffolds from bovine femur
<ol>
	<li>Obtain the condyle from bovine femur from a slaughterhouse certified by health and agricultural authorities of Mexico.
	<ol>
		<li>Carefully dissect the condyle fat, muscle, and cartilage with a surgical instrument. Cut the condyle fragments into 3 cm x3 cm fragments using a saw cutter and clean the fat and blood with a towel. Wash the condyle fragments with water.</li>
		<li>Boil (92 &#176;C) the fragments with 1 L of an anionic detergent (10 g/L) for 30 min. Wash the condyle fragments twice to remove any remnants of the anionic detergent.</li>
		<li>Dry the condyle fragments for 3 h using filter paper (0.5 mm).</li>
	</ol>
	</li>
	<li>Prepare a triangular (1 cm x1 cm x1 cm) CMS of thickness 0.5 cm from the fragments mentioned in step 1.1 (Figure 1A).
	<ol>
		<li>Demineralize the fragments in 100 mL of 0.5 M HCl for 10 min with constant agitation. Remove the HCl. 
		NOTE: Neutralize the HCl with sodium hydroxide (10 M).</li>
		<li>Rinse the fragments three times with 100 mL of distilled water, 15 min each time, with constant agitation. Dry the CMS with filter paper (0.5 mm) for 1 h.</li>
		<li>Use a stereo-microscope15 to analyze the structural properties of the CMS (size of pores, pore formation, and porous interconnection) (Figure 1B).</li>
		<li>Use a scanning electron microscope16 to analyze the rough surface of the CMS trabeculae (Figure 1C).</li>
		<li>Use the dissection instrument for blending and stretching to evaluate the mechanical changes (plasticity and flexibility) of the CMS (Figure 1D).</li>
		<li>Pack the CMS into the sterilization pouch and sterilize it with hydrogen peroxide plasma for 38 min. Store the sterile CMS in the original pack in a dry area at 20-25 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Preparation of the surgical area and handling and preparation of the animal model
<ol>
	<li>Sanitize the surgical area, worktable, microsurgery microscope, and seat with 2% chlorhexidine solution. Sterilize all surgical instruments, surgical sponge, swabs, and disposable surgical drape through heat sterilization (121 &#176;C/30 min/100 kPa)</li>
	<li>Assign the rats (n=5) into three groups of five rats per group: 1. sham, 2. hepatectomy, and 3. hepatectomy plus CMS, and follow all the groups on days 3, 14, and 21 days.
	<ol>
		<li>Administer ketamine (35 mg/kg) and xylazine (2.5 mg/kg) intramuscularly in the hind limb. 
		NOTE: The sedative period typically lasts for 30-40 min.</li>
		<li>Shave the abdominal skin (5 cm x 2 cm) using surgical soap and a double-edged blade, and disinfect the skin using topical 10% povidone-iodine solution in three&#160;rounds17.</li>
		<li>Place the animal on a warm plate in the decubitus dorsal position, with the neck hyperextended to maintain a permeable airway (Figure 2).</li>
		<li>Evaluate the depth of the anesthesia through the respiratory pattern and loss of the withdrawal reflex in the limbs.</li>
		<li>Place a disposable surgical drape around the shaved skin and make an incision (2.5 cm) on the albous line with a scalpel, using the xiphoid process as a reference point. Avoid the abdominal wall blood vessel to prevent bleeding.</li>
		<li>Put the abdominal retractor in place and observe the abdominal cavity. Using the dissection forceps, extract the left liver lobe and place it on the metal plate (Figure 3A). 
		NOTE: In the sham group, only extract the left liver lobe and then return the liver to the abdominal cavity. Suture the abdominal wall and skin with a 3-0 nylon suture.</li>
		<li>In the experimental groups with and without CMS, use a scalpel blade and sterile scalpel blade (#15) to perform hepatectomy (approximately 40%) with two cuts. Use a triangular metallic template (1 cm x 1 cm x 1 cm) to perform the hepatectomy (Figure 3B).</li>
		<li>To prevent bleeding of the liver, maintain surgical compression with a swab on the edge of the liver for 5 min.</li>
		<li>Hydrate the CMS in sterile saline solution for 20 min before the surgical procedure. Implant the CMS in the hepatectomy site with four stitches sutures between the liver tissue and the CMS to prevent displacement of the biomaterial. Use 7-0 non-absorbable polypropylene sutures (Figure 3C). 
		NOTE: Do not remove the sutures in a second surgery; sutures can be used as a reference to identify the site of CMS implantation.</li>
		<li>Return the liver to the abdominal cavity and suture the abdominal wall and skin with a 3-0 nylon suture. Clean the surgical incision with a surgical iodine-soaked sponge in two rounds. Observe and monitor the vital signs of the animals.</li>
	</ol>
	</li>
</ol>
3. Postoperative care
<ol>
	<li>Administer meglumine flunixin (2.5 mg/kg) intramuscularly in the hind limb. Administer analgesics as approved by the institutional animal care and use committee.</li>
	<li>For anesthesia recovery, place the animals in individual polycarbonate boxes with laboratory animal bedding in a noise-free area with temperature control (23 &#176;C).</li>
	<li>Observe the recovery of the animals and monitor their water and food consumption for 2 h. Monitor animals post operatively as approved by the institutional animal care and use committee.</li>
</ol>
4. Evaluation of liver function in serum
<ol>
	<li>Collect blood samples (500 &#181;L) from the lateral tail veins of the anesthetized&#160;animals before the surgical procedure (baseline values) and at evaluation days 3, 14, and 21.</li>
	<li>Centrifuge the blood samples at 850 &#215; g/10 min at room temperature (23 &#176;C); separate the serum and store it at -80&#176;C until use.</li>
	<li>Perform a panel of liver function tests: serum albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and direct bilirubin (DB) (Table 1).</li>
</ol>
5. Euthanasia and tissue management
<ol>
	<li>Anesthetize&#160;the animals for each evaluation (days 3, 14, and 21) by following the protocol described above.</li>
	<li>Euthanize via methods approve by the institutional animal care and use committee.</li>
	<li>Perform an incision (5-6 cm) on the albous line with a scalpel to observe the organs of the abdominal cavity and take photographs of the hepatectomy area of the sham and the experimental groups, with and without the CMS.</li>
	<li>Take liver samples (2 cm x 2 cm; 0.40-0.45 g) from all the study group animals and place them in a 4% formaldehyde solution for 24 h for subsequent histological evaluation.</li>
</ol>
6. Histological analysis
<ol>
	<li>Preserve the liver tissues in 4% formaldehyde and dehydrate the tissue using a series of alcohol concentrations (60%, 70%, 80%, 90%, 100%); place them in xylene (1h) and embedded them in paraffin16.</li>
	<li>Cut the paraffin blocks with a microtome into 4 &#181;m-thick sections for the preparation of eight slides.</li>
	<li>Perform H&#38;E and Masson&#39;s trichrome staining16.</li>
	<li>Observe the stained sections under a light microscope to select the representative areas of the liver, with and without CMS. Obtain photomicrographs at 4x, 10x, and 40x magnification and process the images using appropriate software18&#160;(see the Table of Materials).</li>
</ol>

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The authors declare that they have no competing financial interests.Benjam&#237;n Le&#243;n-Mancilla is a doctoral student from the Programa de Doctorado en Ciencias Biom&#233;dicas, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM) and he received DGAPA-UNAM fellowship.

Organ transplantation is the mainstay of treatment in patients with liver fibrosis or cirrhosis. A few patients benefit from this procedure, making it necessary to provide therapeutic alternatives for patients on the waiting list. Tissue engineering is a promising strategy that employs scaffolds and cells with regenerative potential2,4,13. The removal of a portion of the liver is a critical step in this procedure because of the ...

The liver is one of the most important organs involved in maintaining homeostasis and protein production1. Unfortunately, liver disease is the leading cause of death worldwide. In advanced stages of liver damage, which include cirrhosis and hepatocellular carcinoma, liver transplantation is the clinically recommended procedure. However, due to the scarcity of donors and the low rate of successful transplants, new techniques in tissue engineering (TE) and regenerative medicine (RM) have been developed2,3.
TE involves the use of stem cells, scaffolds, and growth factors4 to promote the restoration of inflamed, fibrotic, and edematous organs and tissues1,5,6. The biomaterials used in scaffolds mimic the native ECM, providing the physical, chemical, and biological cues for guided cellular remodeling7. Collagen is one of the most abundant proteins obtained from the dermis, tendon, intestine, and pericardium8,9. Furthermore, collagen can be obtained as a biopolymer to produce two- and three-dimensional scaffolds through bioprinting or electrospinning10,11. This group is the first to report the use of collagen from a bone source for the regeneration of liver tissue. Another study reports the use of scaffolds synthesized from bovine collagen, which was obtained from skin, with homogeneous and closely situated pores, without any communication between them12.
Decellularization preserves the native ECM, allowing the subsequent incorporation of cells with stem cell potential13,14. However, this procedure is still in the experimental phase in the liver, heart, kidney, small intestine, and urinary bladder from mice, rats, rabbits, pigs, sheep, cattle, and horses3,14. Currently, the resected liver mass volume is not replaced in any of the animal hepatectomy models. However, the use of additional support or network (biomaterials) that enables cell proliferation and angiogenesis could be essential for the prompt restoration of liver parenchymal functions. Thus, scaffolds could be employed as alternative approaches to regenerate or repair tissue in chronic liver diseases, in turn, eliminating limitations due to donation and the clinical complications of liver transplantation.

<table><tbody><tr><td>Anionic detergent</td><td>Alconox</td><td>Z273228</td><td></td></tr><tr><td>Biopsy cassettes</td><td>Leica</td><td>3802453</td><td></td></tr><tr><td>Camera DMX</td><td>Nikon</td><td>DXM1200F</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Chlorhexidine gluconate 4%</td><td>BD</td><td>372412</td><td></td></tr><tr><td>Cover glasses 25 mm x 40 mm</td><td>Corning</td><td>2980-224</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>200-M</td><td>CAS 17372-87-1</td></tr><tr><td>Ethyl alcohol, pure</td><td>Sigma-Aldrich</td><td>459836</td><td>CAS 64-17-5</td></tr><tr><td>Flunixine meglumide</td><td>MSD</td><td>Q-0273-035</td><td></td></tr><tr><td>Glass slides 75 mm x 25 mm</td><td>Corning</td><td>101081022</td><td></td></tr><tr><td>Hematoxylin</td><td>Merck</td><td>H9627</td><td>CAS 571-28-2</td></tr><tr><td>Hydrochloric acid 37%</td><td>Merck</td><td>339253</td><td>CAS 7647-01-0</td></tr><tr><td>Ketamine</td><td>Pisa agropecuaria</td><td>Q-7833-028</td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>Microphoto-FXA</td><td></td></tr><tr><td>Microsurgery stereomicroscope</td><td>Zeiss</td><td>OPMI F170</td><td></td></tr><tr><td>Microtainer yellow cape</td><td>Beckton Dickinson</td><td>365967</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2125</td><td></td></tr><tr><td>Model animal: Wistar rats</td><td>Universidad Nacional Aut&amp;oacute;noma de M&amp;eacute;xico</td><td></td><td></td></tr><tr><td>Nylon 3-0 (Dermalon)</td><td>Covidien</td><td>1750-41</td><td></td></tr><tr><td>Polypropylene 7-0</td><td>Atramat</td><td>SE867/2-60</td><td></td></tr><tr><td>Povidone-iodine10% cutaneous solution</td><td>Diafra SA de CV</td><td>1.37E+86</td><td></td></tr><tr><td>Scanning electronic microscope</td><td>Zeiss</td><td>DSM-950</td><td></td></tr><tr><td>Sodium hydroxide, pellets</td><td>J. T. Baker</td><td>3722-01</td><td>CAS 1310-73-2</td></tr><tr><td>Software ACT-1</td><td>Nikon</td><td>Ver 2.70</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>EZ4Stereo 8X-35X</td><td></td></tr><tr><td>Sterrad 100S</td><td>Johnson and Johnson</td><td>99970</td><td></td></tr><tr><td>Surgipath paraplast</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Synringe of 1 mL with needle (27G x 13 mm)</td><td>SensiMedical</td><td>LAN-078-077</td><td></td></tr><tr><td>Tissue Processor (Histokinette)</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Tissue-Tek TEC 5 (Tissue embedder)</td><td>Sakura Finetek USA</td><td>5229</td><td></td></tr><tr><td>Trichrome stain kit</td><td>Sigma-Aldrich</td><td>HT15</td><td></td></tr><tr><td>Unicell DxC600 Analyzer</td><td>Beckman Coulter</td><td>BC 200-10</td><td></td></tr><tr><td>Xylazine</td><td>Pisa agropecuaria</td><td>Q-7833-099</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>534056</td><td>CAS 1330-20-7</td></tr></tbody></table>

three-dimensional collagen matrix scaffold implantation as a liver regeneration strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Bone demineralization affects the mechanical properties of CMS without altering the original shape or interconnection of its pores. CMS can have any shape, and therefore, can be adjusted to the size and shape of the selected organ or tissue19. In the present protocol, we used a triangular CMS (Figure 1A-D). A rat model was used to evaluate the regenerative capacity of the CMS xenoimplant in the liver. Although the liv...

Watch this Scientific Journal Video about Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy at JoVE.com

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

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JoVE Journal

Medicine

3.0K Views.  Universidad Nacional Autónoma de Mexico (UNAM). Deze methode omvat het plaatsen van een implantaat van collageenmatrix als een steiger in de lever om leverregeneratie te bevorderen. Deze implantatiemethode heeft de deur geopend voor een strateeg die het herstel van de vorm en ballon van de gereseceerde weefsels onderzoekt, waardoor anesthesie, chirurgische duur en hersteltijd worden verminderd. Deze methode kan worden gebruikt om regeneratie te bevorderen met modellen van leverfibrose, evenals andere zoogdiersoorten.Deze methode is ...

Video: Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Bioengineering

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Cancer Research

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

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.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

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.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

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

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

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.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

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