We wish to thank Dr. Rossant and Dr. Bilodeau for the Nkx2-1mcherry ESC used in experiments depicted in Figures 1-3. FACS was performed in The SickKids-UHN Flow Cytometry Facility. This work was supported by operating grants from the Canadian Institutes for Health Research and an infrastructure grant (CSCCD) from the Canadian Foundation of Innovation.

Animal experiments were carried out in accordance with the Animal Care Committee guidelines of the Hospital for Sick Children Research Institute.
1. Scaffold Preparation
<ol>
	<li>Decellularization of lungs
	<ol>
		<li>Euthanize adult Wistar rats using CO2 chamber. Place animal in the chamber and start 100% CO2 exposure at a fill rate of 10-30% of chamber volume per minute.
		<ol>
			<li>Observe animal for unconsciousness; this will occur after approximately 2-3 min. If unconsciousness does not occur in this time period, check fill rate and chamber seal. Following unconsciousness, observe animal for faded eye color and lack of respiration. If both are observed, maintain CO2 filling for 1-2 min and then remove animal from chamber for procedure.</li>
		</ol>
		</li>
		<li>Secure animal to dissecting surface by fixing forepaws and hind legs, and spray down the chest and abdomen area with 70% ethanol. Access the heart and lungs by opening the thoracic cavity using a vertical incision along the sternum.</li>
		<li>Make small incisions through diaphragm first to cause the lungs to retract, reducing the chance of puncturing the lungs. Ligate the inferior vena cava with a suture and place a small incision in the left atrium with small dissecting scissors.</li>
		<li>Using a prepared 10 ml syringe with a 25 G needle filled with heparinized Hank&#8217;s balanced salt solution (HBSS) (10 U/ml heparin in HBSS), start lung perfusion by inserting the needle into the right ventricle to push the buffer through the pulmonary circulation (rate of 2 ml/min). Continue this procedure until the lungs turn white and the fluid flowing from the left atrium runs clear.</li>
		<li>Following perfusion, expose the trachea and cannulate with a plastic catheter near the thyroid cartilage and secure in place with a suture.</li>
		<li>Setup a gravity perfusion system by fixing a 10 ml syringe to a retort stand and clamp. Remove and discard syringe plunger. Secure the maximum filling point on syringe barrel at 20 cm above the lungs. Attach a two-way stopcock to the end of the syringe, and a long plastic tubing to the other end of the stopcock for delivering decellularization solution to the cannulated trachea. Pour solution into syringe and allow solution to fill attached plastic tubing and catheter.
		<ol>
			<li>Lavage the lungs by filling to total lung capacity (approximately 12 ml) for 1 min and remove the plastic catheter from the trachea to allow the fluid to flow out of the lungs. Do not fill syringe more than 10 ml when lavaging lungs to keep pressure below 20 cm of H2O.</li>
		</ol>
		</li>
		<li>Repeat lavage of lungs eight times with decellularization solution, followed by 10 rinses with phosphate-buffered saline (PBS).</li>
		<li>Dissect the trachea and lungs free from the neck and chest cavity and remove from the animal. Keep tissue in cold PBS at 4 &#176;C until preparation for vibratome sectioning.</li>
	</ol>
	</li>
	<li>Thick section generation
	<ol>
		<li>Prepare approximately 15 ml of 2% and 4% (w/v) agarose, and enough 6% (w/v) agarose for embedding all lobes into small rectangular blocks. Dissolve low melting point agarose powder in PBS by microwaving. Transfer the agarose to 50 ml tubes on a heat block and maintain temperature above 40 &#176;C to avoid gelling.</li>
		<li>Dissect decellularized lung at the end of each lobar bronchus to detach each lobe (cranial, middle, accessory, and caudal right lobes and the left lobe) using small scissors. Pat dry each lobe using absorbent sheets to remove excess PBS and place inside 2% (w/v) agarose while on the heating block.</li>
		<li>Remove each lobe after 5 min of coating in agarose, place in a Petri dish and allow the surface to gel for 1 min on a cold plate.</li>
		<li>Gently place lobes back into 4% (w/v) agarose, cool after 5 min, and repeat coating once more with the 6% (w/v) agarose.</li>
		<li>After sequential coating of each lobe, embed each lobe separately in 6% (w/v) agarose using metal base molds with at least 3 mm of agarose surrounding the tissue from the edges. Orient each lobe using forceps by positioning the lobe&#39;s largest flat edge at the surface of metal mold facing the experimenter. This edge will be the side fixed to the specimen plate for vibratome sectioning.</li>
		<li>Allow blocks to gel on cold plate for at least 30 min prior to sectioning with vibratome. Store blocks in a humidified chamber for up to 12 hr at 4 &#176;C prior to vibratome sectioning.</li>
		<li>Setup the vibratome by filling the sectioning chamber with cold PBS12. Maintain cold temperature throughout sectioning with the surrounding ice bath. Remove blocks from metal molds and use a razor blade to trim down excess agarose surrounding lobes, while keeping approximately 3 mm from the edge of the tissue.</li>
		<li>Fix tissue to the center of specimen plate using adhesive, and submerge plate into the PBS-filled sectioning chamber. Setup sectioning boundaries on vibratome by selecting the following speed, amplitude, and thickness values respectively: 0.2 mm/sec, 1.85 mm, and 350 &#181;m. 
		Note: Both longitudinal and transverse sections of the lobe orientation are acceptable.</li>
		<li>Section each lobe completely. Manually cut sections free using small scissors if a section is not fully separated by blade at the end of the section sequence. Collect scaffold sections gently and keep in PBS on ice until the next step. 
		Note: Sections generated will include both the proximal and distal lung areas and both sources can be used for recellularization; however, most of the surface will encompass distal lung.</li>
	</ol>
	</li>
	<li>Decontamination of scaffold sections
	<ol>
		<li>Transfer the 350 micron thick scaffold sections from PBS to microcentrifuge tubes (up to 30 sections/tube) and treat with nuclease (90 U/ml in PBS) (see List of Materials) for 12 - 24 hr at RT, on a rotator.</li>
		<li>Following nuclease treatment, transfer sections using forceps to new microcentrifuge tubes and treat with antimicrobial solution (200 U/ml penicillin streptomycin and 25 &#181;g/ml amphotericin B in PBS) under sterile conditions for 6 hr at RT, on a rotator. 
		Note: Scaffolds can be stored in antimicrobial solution for up to one week at 4 &#176;C before use.</li>
		<li>Following the decontamination step with antimicrobial solution, rinse scaffolds twice with PBS under sterile conditions and transfer to serum free differentiation media (SFDM) prior to seeding with cells.</li>
	</ol>
	</li>
</ol>
2. Endodermal Cell Preparation
<ol>
	<li>Definitive Endoderm Induction
	<ol>
		<li>Maintain mouse ESC lines under feeder-free, serum free culture using 2i conditions13. Start endoderm induction by removing cells from adherent pluripotent culture by trypsinization.</li>
		<li>Resuspend cells in SFDM and seed at a density of 20,000 cells/ml in low adherent plates for three days without media change to allow for EB formation.</li>
		<li>After three days gently transfer the EBs into 50 ml conical tubes using a 10 ml pipette and allow them to collect at the bottom for 3 min at RT.</li>
		<li>Carefully aspirate media and add fresh SFDM media supplemented with 50 ng/ml activin A.</li>
		<li>Seed cells back onto the low adherent plates at a 1:2 density and culture for three additional days to achieve definitive endoderm differentiation.</li>
	</ol>
	</li>
	<li>Definitive endoderm enrichment
	<ol>
		<li>Collect day 6 EBs, dissociate with trypsin and label for c-KIT and CXCR4 expression using conjugated fluorescent antibodies14.</li>
		<li>Sort labeled cells using fluorescence-activated cell sorting (FACS) for expression of both markers to obtain an enriched definitive endoderm population15.</li>
	</ol>
	</li>
</ol>
3. Recellularization Setup
<ol>
	<li>Air-liquid interface culture setup
	<ol>
		<li>Transfer each decellularized scaffold section (step 1.2.9) from SFDM onto a hydrophobic floating membrane (8 &#181;m pore size) using sterile forceps. Ensure scaffold sections are spread evenly on membrane.</li>
		<li>Prepare 6- or 12-well plates by filling wells with 1 or 0.5 ml of SFDM, respectively. Gently place membranes into wells, allowing the membrane to float on top of media, creating an air-liquid culture setup.</li>
	</ol>
	</li>
	<li>Seeding of 3D scaffolds
	<ol>
		<li>Following FACS for definitive endoderm markers (step 2.2.2) count sorted cells using a hemocytometer, spin down at 400 x g for 5 min and resuspend in SFDM. Resuspend cells to obtain a volume containing approximately 100,000 cells/10 &#181;l/scaffold.</li>
		<li>To recellularize scaffolds, pipette 10 &#181;l of cells directly onto each prepared section from step 3.1.2.</li>
		<li>Replace SFDM media in cultures every 48 hr. Aspirate the old media by holding the plate at a slight incline to avoid disrupting the culture. Slowly add fresh SFDM to culture along the side of the well to prevent sinking of floating membrane.</li>
		<li>Maintain air-liquid cultures for up to 21 days to achieve differentiation of seeded cells in to mature airway epithelia. 
		Note: Recellularized sections can be processed for tissue staining and immunofluorescent (IF) microscopy at any time point during cell culture16.</li>
	</ol>
	</li>
</ol>

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There are no competing financial interests to declare.

The protocol described here generates mature ESC-derived airway epithelia using only natural lung scaffolds to direct differentiation with no other supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. No growth factor supplementation of base differentiation media is required. Previously published methods for generating stem cell-derived lung epithelial cells have used 2-dimensional strategies with growth factor supplementation to promote lineage restriction3,4,8,18,19. The technique described here is advantageous over these methods for several reasons, including: defined culture conditions, high differentiation efficiency to functional airway cells, limited contamination from other endodermal lineages (thyroid, liver, pancreas), and a 3D differentiation microenvironment. This technique yields similar results with mouse-derived ECM and can therefore be modified to generate decellularized scaffolds using mouse lungs. Rat-derived ESCs differentiated to definitive endoderm and seeded onto scaffolds using this protocol also differentiate to airway epithelia.
Decellularized scaffolds can be stored in decontamination solution at 4 &#176;C for up to 7 days prior to recellularization. Scaffolds kept for longer durations may be functional, however we have not tested to confirm this. To achieve efficient organization and differentiation it is required to enrich for definitive endoderm by FACS following induction with activin A. Non-endoderm cells in day 6 EBs often express pluripotency marker Oct4 and if seeded on scaffolds will continue to proliferate without organization and specification to the lung lineage. The seeding density can also be adjusted based on cell line-specificity to achieve complete recellularization of scaffolds.
A limitation of this protocol is in its inability to promote differentiation to alveolar epithelial cells. Although NKX2-1+/SOX9+ distal progenitor cells are detected after seven days of culture on scaffolds, this population is not present at day 14, and day 21. Markers of mature type I alveolar epithelial cells (AQP5) and type II alveolar epithelial cells (SFTPB, SFTPC) are not detected at any stage during scaffold culture. This may be due to a requirement for additional growth factor supplementation and submerged culture conditions to promote alveolar lineage specification, while as described here, matrix proteins and the remnant matrix-bound growth factors on decellularized scaffolds is sufficient to promote airway lineage differentiation.
The critical step for promoting differentiation of definitive endoderm to airway epithelial cells using lung scaffolds is optimization of the decellularization process. Cellular components must be completely removed while matrix components are preserved. Tissue staining for nuclear material using DAPI, ultra-structural analysis of scaffolds with scanning and transmission electron microscopy, and DNA assays can be used to confirm removal of all cellular components. ECM protein composition of scaffolds can be assessed using IF staining and immunoblot analysis for matrix proteins: collagen I, collagen IV, elastin, fibronectin, heparin sulfate proteoglycans, and laminin16.
These stem cell-derived lung epithelia can be used in disease modeling and drug-discovery platforms of airway-related pathologies such as cystic fibrosis. The potential of these cell-matrix constructs in regenerative applications such as tissue repair and cell therapy can be examined by in vivo transplantation of engineered constructs in various mouse models. Furthermore, this 3D in vitro method for airway differentiation can be easily adapted to study various parameters effecting lung lineage specification by manipulating growth factor availability and cell-matrix signaling of cultures during different stages of differentiation on scaffolds.

Directed differentiation of pluripotent cells to the lung lineage is dependent on precise signaling events in the microenvironment 1,2. Due to the dynamic nature of this process it has been challenging to mimic the precise events of lung organogenesis in vitro. Recent reports have used step-wise lineage restriction strategies with soluble growth factor supplementation of two-dimensional cultures to achieve lung differentiation3-8. In step-wise differentiation protocols, pluripotent cells, whether embryonic stem cells (ESC) or induced pluripotent stem cells, were first differentiated to the definitive endoderm germ layer. Endodermal cells were subsequently pushed to an anterior endoderm fate and thereafter to lung progenitor cells, as identified by the expression of homeodomain-containing transcription factor NKX2-1. These lung progenitors were further differentiated to proximal (airway) or distal (alveolar) lung epithelial cells with continued growth factor supplementation. Such 2-dimensional strategies have had some success in generating lung epithelial cells, however there are several limitations including unclear efficiencies, possible contamination from other endodermal lineages, lack of a 3-dimensional (3D) structure, and in some instances use of undefined cultures with serum supplementation. Culture of pluripotent or differentiated cells on decellularized lung scaffolds is increasingly used as an assay to assess the regenerative potential of seeded cells in forming lung epithelial structures3,5,6,8,9. Such reports culture seeded cells on scaffolds with continued growth factor or serum supplementation.
Lung development involves the division, migration, gene expression and differentiation of individual cells in response to environmental cues. The extra cellular matrix (ECM) is a latticework of glycoproteins that in addition to providing structural support, directs tissue morphogenesis by integrating and regulating these processes10,11. By using the lung ECM scaffold as a natural platform for endoderm culture to better mimic the in vivo lung developmental milieu, we have generated stem cell-derived airway epithelial cells in a defined 3D-culture setting with high efficiency and reproducibility.
Rat lung ECM scaffolds were generated by decellularization as well as mouse ESC-derived endodermal cells were generated and subsequently seeded onto these scaffolds. Dual expression of CXCR4 &#38; c-KIT proteins indicates a definitive endoderm cell identity and cells positive for both SOX2 &#38; NKX2-1 expression are identified as airway (proximal lung) progenitor cells. Definitive endoderm cells were cultured at air liquid interface (ALI) for up to three weeks to generate functional airway epithelial cells in vitro.
This protocol promotes lung lineage differentiation of definitive endoderm as early as 7 days, observed with the emergence of NKX2-1+/SOX2+ early proximal lung progenitors. By day 14 and 21 of culture mature airway epithelial cell populations emerge including ciliated (TUBB4A+), club (SCGB1A1+), and basal (TRP63+, KRT5+) cells with morphological and functional resemblance to native mouse airways. This protocol demonstrates the importance of the 3D-matrix microenvironment for achieving robust differentiation to airway epithelial cells.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Reagents</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">H0777</td>
			<td style="width:281px;">10U/mL heparin</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">14170112</td>
			<td style="width:281px;">dissolved in Hank&#39;s balanced salt solution (HBSS-)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">CHA003</td>
			<td style="width:281px;">8mM CHAPS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">E9884</td>
			<td style="width:281px;">25mM EDTA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">SOD002</td>
			<td style="width:281px;">1M NaCl</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">dissolved in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Novagen</td>
			<td style="width:168px;">70664-3</td>
			<td style="width:281px;">90U/mL Benzonase nuclease&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15140</td>
			<td style="width:281px;">200U/mL penicillin streptomycin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15290</td>
			<td style="width:281px;">25&#956;g/mL amphotericin B&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Trypsinization&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12605-028</td>
			<td style="width:281px;">TrypLE</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">IMDM 2440-053, F12 11765-054</td>
			<td style="width:281px;">3:1 ratio of IMDM and Ham&#8217;s modified F12 medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12587-010</td>
			<td style="width:281px;">B27 supplement (50x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">17502-048</td>
			<td style="width:281px;">N2 supplement (100x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">15260-037</td>
			<td style="width:281px;">0.05% (Fraction V) bovine serum albumin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">35050-061</td>
			<td style="width:281px;">200mM Glutamax&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">M6145</td>
			<td style="width:281px;">4&#956;M monothioglycerol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">A4403&#160;</td>
			<td style="width:281px;">0.05mg/mL ascobic acid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Endoderm induction</td>
			<td style="width:163px;">R&#38;D</td>
			<td style="width:168px;">338-AC/CF</td>
			<td style="width:281px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antibodies</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CDH1</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">610181</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">C-KIT</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558163</td>
			<td style="width:281px;">Rat, PE-Cy7, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CXCR4</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558644</td>
			<td style="width:281px;">Rat, APC, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">KRT5</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab24647</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">NKX2-1</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab76013</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Laminin</td>
			<td style="width:163px;">Novus Biologicals</td>
			<td style="width:168px;">NB300-144</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SCGB1A1</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-9772&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SOX2</td>
			<td style="width:163px;">R&#38;D Systems</td>
			<td style="width:168px;">AF2018&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TRP63</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-8431</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TUBB4A</td>
			<td style="width:163px;">BioGenex</td>
			<td style="width:168px;">MU178-UC</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Goat IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-11055</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21202</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31571</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21206</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31573</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Other Materials</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low adherent plates</td>
			<td style="width:163px;">Nunc</td>
			<td style="width:168px;">Z721050&#160;</td>
			<td style="width:281px;">Low cell binding plates,&#160; 6 wells&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air-liquid interface membranes&#160;</td>
			<td>Whatman</td>
			<td align="right">110614</td>
			<td>Hydrophobic Nucleopore membrane, 8&#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1200S&#160;</td>
			<td>Leica Vibratome</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tissue Adhesive</td>
			<td>Ted Pella</td>
			<td align="right">10033</td>
			<td>Pelco tissue adhesive</td>
		</tr>
	</tbody>
</table>

generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

As outlined in this protocol, robust differentiation of definitive endoderm to mature airway epithelial cells can be achieved using extended culture of seeded cells on decellularized lung scaffold sections. It is recommended that decellularized scaffolds be characterized to ensure (1) host cells are completely removed, and (2) extracellular matrix proteins are preserved prior to using scaffolds for differentiation. Decellularization can be assessed using tissue staining with hematoxylin and eosin (H&#38;E) and 4&#39;,6-diamidino-2-phenylindole (DAPI), as illustrated in Figure 1A. High-magnification scanning electron microscope (SEM) analysis of scaffolds confirms the absence of host cells and intact matrix architecture, as illustrated in Figure 1B. Additional characterization of the remaining scaffold by tensile testing and immunostaining for matrix proteins can be carried out to ensure preservation of the ECM following decellularization16.
Non-adherent culture of ESCs in SFDM media for six days results in the formation of cell aggregates (EBs) as illustrated in Figure 2A. Three days of activin A treatment results in definitive endoderm induction. Lineage restriction of ESC to definitive endoderm can be confirmed by upregulation and expression of endoderm lineage-associated genes and proteins17, and cannot be asserted morphologically. Endoderm-induction efficiency ranges from 50-70% depending on the ESC line used. Experiments were carried out in mouse ESC lines: R1 (Nkx2-1mCherry), G4 (dsRed-MST), and 129/Ola (Bry-GFP/Foxa2-hCD4), while all data illustrated in this manuscript is using the R1 cell line. The double positive CXCR4+/c-KIT+ definitive endoderm population is sorted as illustrated in Figure 2B, seeded onto 3D lung scaffolds, and cultured for up to 21 days at air-liquid interface in SFDM. It is important to sort and enrich for definitive endoderm as limited organization and differentiation is achieved with culture of unsorted cells from heterogeneous day 6 EBs as illustrated in Figure 2C. Structures formed by sorted cells on scaffolds are reminiscent of developing lung (embryonic day 13.5) as illustrated in Figure 2C-D. In our experience, a seeding density of 100,000 sorted cells/scaffold has yielded the best scaffold repopulation.
Differentiation to the lung lineage is observed as early as 7 days following scaffold culture. Airway epithelial progenitor cells positive for NKX2-1 and SOX2 are detected using IF confocal analysis as illustrated in Figure 3A. SOX2 positive, NKX2-1 negative cells are likely pluripotent endodermal cells that have not differentiated to the lung lineage. With longer culture these cells may start to express NKX2-1 or undergo apoptosis without adequate support in the microenvironment. IF analysis of day 21 cultures reveals a mature airway epithelial population containing TUBB4A+ ciliated cells, SCGB1A1+ club cells, and TRP63+/KRT5+ basal cells as illustrated in Figure 3B. SEM analysis of the surface of day 21 cultures reveals morphological similarity of stem cell-derived cultures to mouse airways.
Both isolated matrix proteins (collagen I, collagen IV, fibronectin, laminin) and decellularized rat kidney scaffolds were unable to promote lung-lineage differentiation when seeded with definitive endoderm and cultured under the same conditions as described above16. This demonstrates that the 3D microenvironment created by lung-derived scaffolds is distinct from kidney-derived scaffolds in its ability to promote lung-lineage differentiation of seeded endodermal cells.
<img alt="Figure 1" src="/files/ftp_upload/54019/54019fig1.jpg" /> 
Figure 1. Decellularization technique removes cellular components and preserves ECM. (A) H&#38;E (top panel) and DAPI (lower panel) staining of natural and decellularized lung tissue showing the absence of nuclei following decellularization. Right panel shows examples of tissue sections from lungs without optimal decellularization. Arrowheads show remaining nuclei on scaffolds due to incomplete decellularization, highlighting the importance of optimizing technique before recellularization. Scale bar = 25 &#181;m. (B) SEM images of natural and decellularized lung, showing absence of cells and preservation of matrix architecture on decellularized scaffolds. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54019/54019fig2.jpg" /> 
Figure 2. Sorted ESC-derived definitive endoderm results in optimal recellularization. (A) Day 6 embryoid bodies (EBs) following three days of activin A treatment for promoting definitive endoderm differentiation. (B) The EBs are sorted by FACS for dual expression of surface markers CXCR4 and c-KIT to isolate the definitive endoderm population. This population will be seeded on decellularized scaffolds. (C) H&#38;E staining of recellularized scaffolds at day 7 and day 21 of culture. Left panel represents organized, airway-like structures following seeding with sorted cells, and right panel represents disorganized, highly proliferative unsorted cells on scaffolds highlighting the importance of sorting of cells prior to recellularization. Scale bar = 30 &#181;m. (D) H&#38;E staining of embryonic day 13.5 mouse lungs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54019/54019fig3.jpg" /> 
Figure 3. Extended culture of definitive endoderm on natural lung scaffolds directs differentiation to airway epithelial cells. (A) Proximal lung progenitor cells (NKX2-1+/SOX2+) are detected following seven days of culture on decellularized scaffolds. Scale bar = 15 &#181;m. (B) Left image shows mature pseudostratified epithelial cell (CDH1+) structures lined by basement membrane protein laminin on the basal side of structures. Center and right image show mature airway epithelia complete with ciliated cells (TUBB4A+), club cells (SCGB1A1+) and basal cells (KRT5+/TRP63+). Left image scale bar = 30 &#181;m; center and right image scale bar = 15 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.6K Views.  Hospital for Sick Children. This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Video: Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...