SH acknowledges support from the Institute Post-Doctoral Fellowship of IIT Kanpur and SYST grant from DST (SEED Division) (SP/YO/618/2018). AM acknowledges the Indian Institute of Technology-Kanpur (IIT-Kanpur) for an Institute fellowship. DSK acknowledges Gireesh Jankinath Chair Professorship and Department of Biotechnology, India, for funding (BT/PR22445/MED/32/571/2016). AM, SH, and DSK thank The Mehta Family Centre for Engineering in Medicine at IIT-Kanpur for their generous support.

The protocol is based on the isolation of IFP-MSCs from goats. Goat IFP and blood were collected from a local abattoir. Since such tissue collections are outside the purview of an Institutional Animal Ethics Committee, ethical approval was not required.
1. Isolation of IFP-MSCs from the femorotibial joint of goat knee
<ol>
	<li>Collect goat femorotibial joint (sample) encompassing ~15 cm each of the femoral and tibial regions of the hind limbs. Immediately place the sample in a sterilized sample collection box and store it at 4 &#176;C for transportation to the laboratory for further processing. Process the samples aseptically in a biosafety cabinet, using sterilized surgical tools throughout the procedure (Figure 1, Step 1).</li>
	<li>Place the sample on a 150 mm Petri dish and rinse it thoroughly with autoclaved phosphate-buffered saline (PBS) supplemented with antibiotics (5 &#181;g/mL amphotericin B, 200 U/mL penicillin-streptomycin, and 50 &#181;g/mL ciprofloxacin) to avoid contamination. Always ensure that the sample is kept hydrated using PBS.</li>
	<li>Carefully examine the anatomical regions of the sample. For ease of access to the joint capsule, ensure that the extra tissues from both the long bones are completely removed (Figure 1, Step 2).</li>
	<li>To achieve this, first, hold the end surface of the femur bone with one hand and, with sharp scissors, cut longitudinally towards the joint. Remove the surrounding muscles and adipose tissue from the bone during the process. Be careful that the tissue surrounding the immediate joint capsule remains undisturbed initially.</li>
	<li>Similarly, make another incision at the tibial end of the sample and remove the muscles and adipose tissue from the tibial bone. Ensure that both the long bones are completely exposed and the joint capsule is clearly visible after this step (Figure 1, Step 3).</li>
	<li>Next, make an incision from either side of the synovium membrane to cut open the articulating joint (Figure 1, Step 4). Cut free the patella from both the synovium membrane and patellar ligaments using a fresh batch of sterilized scissors and forceps. Immediately keep the separated patella in a Petri dish containing PBS (Figure 1, Step 5).</li>
	<li>Remove the entire fat pad present on the inner surface of the patella using scissors and forceps and collect the fat pad in another fresh Petri dish (Figure 1, Step 6). Mince the collected fat pad using a scalpel. Include other surgical tools (e.g., scissors or surgical blades) as required to obtain the smallest possible fat pieces/segments of ~2-3 mm. 
	NOTE: Good mincing is critical to result in a good yield of cells. Always keep the tissue hydrated and do not let it dry at any stage of the isolation procedure.</li>
	<li>Transfer the minced tissue using a spatula to a 50 mL centrifuge tube and wash it with PBS supplemented with antibiotics at room temperature (RT) for 15 min in a test tube mixer. Repeat the washing step 3x, 15 min each.</li>
	<li>For enzymatic digestion, incubate the minced tissue (~5 g) in Dulbecco&#39;s Modified Eagle&#39;s Media (DMEM low glucose) containing 1.5 mg/mL type II collagenase (20 mL) and supplemented with 2% FBS at 37 &#176;C for 12-16 h. 
	NOTE: Perform the digestion in a test tube mixer at ~15 rotations per minute (RPM).</li>
	<li>Carefully aspirate the digested tissue and filter it through a cell strainer (70 &#181;m) to remove any undigested tissue. Centrifuge the filtrate in a 15 mL centrifuge tube at 150 x g for 5 min. Remove the supernatant and wash the pellet with DMEM low glucose at least 2x. 
	NOTE: Pour the digested tissue slowly into the strainer to avoid spillage. Use a low volume centrifuge tube (15 mL) to get a good pellet.</li>
	<li>Resuspend the obtained cell pellet in complete DMEM media (DMEM low glucose supplemented with 10% FBS and antibiotics [2.5 &#181;g/mL amphotericin, 100 U/mL penicillin-streptomycin, and 25 &#181;g/mL ciprofloxacin]), referred to as expansion media in the protocol in the subsequent steps.</li>
	<li>Seed the cells on 150 mm Petri dishes and culture them in the expansion media supplemented with 5 ng/mL basic fibroblast growth factor (bFGF) and 50 &#181;g/mL 2-Phospho-L-ascorbic acid trisodium salt. Incubate the cells at 37 &#176;C in a 5% CO2 incubator to allow the cells to adhere and proliferate. Monitor the attachment and growth of the cells on a daily basis.</li>
</ol>
2. Maintenance and expansion of isolated cells
<ol>
	<li>Change the media every 3 days until the cells become confluent. Add fresh 5 ng/mL bFGF and 50 &#181;g/mL 2-Phospho-L-ascorbic acid trisodium salt directly to the Petri dish every time the media is changed. After the cells become 80%-90% confluent, subculture the cells. 
	NOTE: Remove the non-adherent entities during each media change. In case oil droplets are seen after 3 days of incubation, wash the cells with PBS 1x and then add fresh media to the Petri dish. Continue with PBS wash before each media change until the oil droplets are completely removed.</li>
	<li>Sub-culturing of cells: Remove the expansion media from the Petri dish and wash the cells 1x with PBS. Add 4 mL of 0.25% trypsin/EDTA to the dish and incubate the cells at 37 &#176;C in a 5% CO2 incubator for 4 min. 
	NOTE: Confluency (80%-90%) is achieved within 7 days. Uniformly distribute the trypsin/EDTA solution over the entire surface of the Petri dish during trypsinization.</li>
	<li>Dislodge the cells by tapping the plate on the side and observing under a microscope to confirm that all the cells are detached. Neutralize the trypsin after adding an equal volume (4 mL) of expansion media.</li>
	<li>Collect the dissociated cells using a pipette in a fresh 15 mL polypropylene tube and centrifuge at 150 x g for 5 min at RT. Discard the supernatant and resuspend the pellet in the desired volume of expansion media.</li>
	<li>Count the cells using the standard cell counting method and subculture in 150 mm Petri dishes at a seeding density of 1 x 104 cells/cm2 for further expansion. For all assays, use a confluent monolayer of cells of passage number P2-P5. 
	&#8203;NOTE: Cryopreserve the excess cells.</li>
</ol>
3. Evaluation of the clonogenic ability of IFP-MSCs using colony forming assay (CFU-F)
<ol>
	<li>For CFU-F assay, seed the cells in expansion media in a 6-well tissue culture plate at a seeding density of 50-100 cells/cm2. Add media to obtain a final volume of 3 mL. 
	NOTE: Optimize the cell concentration for assays and treatment.</li>
	<li>Culture the cells for 12 days at 37 &#176;C in a 5% CO2 incubator under standard culture conditions to allow the cells to form colonies. Change the media every 3 days. Be gentle while changing the media.</li>
	<li>After 12 days, rinse the colonies 1x with PBS and fix them using 20% methanol for 20 min at RT. Stain the colonies with crystal violet dye (0.5% [w/v] in 20% methanol) for 20 min at RT. Count the individual colonies using an inverted microscope. 
	&#8203;NOTE: A colony is defined as a cluster of more than 50 cells.</li>
</ol>
4. Differentiation potential of IFP-MSCs
<ol>
	<li>Inducing adipogenic differentiation of IFP-MSCs
	<ol>
		<li>To induce adipogenesis, seed the cells at a density of 25 x 103 cells/cm2 in a 24-well tissue culture plate. Add media to obtain a final volume of 500 &#181;L. Culture the cells at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>One day post-confluency, replace the expansion medium with 500 &#181;L of adipogenic differentiation media. It requires approximately 2 days for the cells to reach confluency.</li>
		<li>Prepare the adipogenic differentiation media by supplementing expansion media (DMEM + 10% FBS + 2.5 &#181;g/mL amphotericin, 100 U/mL penicillin-streptomycin, and 25 &#181;g/mL ciprofloxacin) with 25 mM D (+)-glucose, 10 &#181;g/mL insulin, 1 &#181;M dexamethasone, and 100 &#181;M indomethacin. 
		NOTE: Differentiation media is unstable at 4 &#176;C after 1 month; hence, prepare the media as and when required.</li>
		<li>Consider the cells cultured in the expansion media supplemented with 25 mM D (+) glucose as uninduced controls. Change the media every 3 days for 14 days. At the end of the 14 days, wash the cells 1x with PBS. Fix the cells using neutral buffered formalin (NBF; 4 % formaldehyde in PBS) for 15 min at 4 &#176;C.</li>
		<li>Following fixation, wash the wells 1x with distilled water and then incubate the cells with 60% isopropanol for 5 min at RT. After removing isopropanol, stain the lipid droplets by incubating the cells with 500 &#181;L of the working solution of Oil Red O dye for 5 min at RT. Wash the wells 1x with distilled water to remove the unbound dye and image the cells using an inverted microscope. 
		NOTE: Prepare the stock solution of Oil Red O (ORO) by dissolving 300 mg of ORO in 100 mL of 99% isopropanol. To prepare the working solution, mix three parts of stock ORO and two parts of distilled water and allow it to mix at RT for 10 min. Filter the mixture using 0.2 &#181;m filter paper. The working solution is ready to use. The stock solution can be prepared and stored at RT for 1 month in the dark, whereas the working solution needs to be freshly prepared prior to each use/staining.</li>
	</ol>
	</li>
	<li>Inducing chondrogenic differentiation of IFP MSCs
	<ol>
		<li>Isolation of goat plasma:
		<ol>
			<li>Prepare 3.4% (w/v) sodium-citrate in distilled water. Add 5 mL of the prepared sodium citrate solution to a 50 mL centrifuge tube.</li>
			<li>Collect 45 mL of the freshly isolated goat blood in the same tube. Immediately tilt the tube to mix the blood with sodium citrate thoroughly to prevent clotting and transport it to the laboratory at 4 &#176;C.</li>
			<li>Centrifuge the collected goat blood at 1000 x g for 20 min at 4 &#176;C. Transfer the supernatant (plasma) to a fresh tube inside a biosafety cabinet. Filter the plasma through 0.2 &#181;m filters, aliquot, and store at -20 &#176;C for further use. 
			NOTE: Maintain the temperature of 2-8 &#176;C while handling the samples. It is important to minimize the freeze-thaw cycles of plasma.</li>
		</ol>
		</li>
		<li>Culture the expanded cells to 80%-90% confluency, dissociate the cells using trypsin/EDTA solution, and pellet down the cells at 150 x g for 5 min in a 15 mL centrifuge tube. Resuspend the pellet in goat plasma at a density of 2 x 106 cells per 50 &#181;L of plasma solution.</li>
		<li>Add a drop of plasma-containing cells (50 &#181;L) onto a sterilized 90 mm Petri dish and then add calcium chloride at a final concentration of 0.3% (w/v). Mix well to fabricate cross-linked plasma hydrogel.</li>
		<li>Incubate the fabricated hydrogel at 37 &#176;C for 40 min. After incubation, transfer the hydrogels to 24-well tissue culture plates containing chondrogenic media.</li>
		<li>Prepare chondrogenic media by supplementing DMEM low glucose with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.25 mg/mL bovine serum albumin (BSA), 1 mM proline, 50 &#181;g/mL 2-Phospho-L-ascorbic acid trisodium salt, 100 nM dexamethasone, 1x insulin, transferrin, sodium selenite + linoleic-BSA (ITS+1), antibiotics (2.5 &#181;g/mL amphotericin, 100 U/mL penicillin-streptomycin and 25 &#181;g/mL ciprofloxacin), and 10 ng/mL transforming growth factor- &#946;1 (TGF-&#946;1).</li>
		<li>The hydrogels cultured in complete DMEM media (DMEM low glucose with 10% FBS and antibiotics [2.5 &#181;g/mL amphotericin, 100 U/mL penicillin-streptomycin, and 25 &#181;g/mL ciprofloxacin]) without TGF-&#946;1 are considered as uninduced controls. Change the media every 3 days for 14 days.</li>
		<li>After 14 days of chondrogenic differentiation of cells in plasma hydrogels, wash the hydrogels with PBS 3x for 5 min each and then fix the samples in NBF for 3 h. 
		CAUTION: Formaldehyde is a potential carcinogen and can cause irritation to the nose, throat, and lungs upon inhalation. Perform all procedures related to formaldehyde in a fume hood.</li>
		<li>Remove NBF, wash the hydrogels with PBS 3x for 5 min each, and then incubate in 35% (w/v) sucrose at RT overnight to allow the solution to be completely absorbed into the samples. Acclimatize the hydrogels in optimal cutting temperature (OCT) compound for 3 h. Embed the samples in OCT compound using silicone molds under liquid nitrogen.</li>
		<li>Cut the samples at a thickness of 10-12 &#181;m on gelatin-coated glass slides using a cryotome and store them at -20 &#176;C for future use. To examine the presence or deposition of the extracellular matrix after chondrogenic differentiation, stain the sections with Alcian Blue and Safranin-O dye, which bind to sulfated glycosaminoglycans. 
		NOTE: To prepare Alcian Blue dye, dissolve 1 g of Alcian Blue dye in 100 mL of 0.1 N HCl and mix thoroughly in a test tube mixer overnight. The solution can be stored at RT for 1 month in the dark. To prepare Safranin-O dye, dissolve 0.1 g of Safranin-O dye in 100 mL of distilled water and mix thoroughly in a test tube mixer overnight.</li>
		<li>For Alcian Blue staining, wash the hydrogel sections 1x with distilled water for 5 min. To fix the sections, add 4% NBF to the slides and incubate for 30 min.</li>
		<li>Wash the sections 1x with distilled water for 5 min and then 2x with 0.1 N HCl for 30 s each. Remove the HCl and gently dry the slides with tissue paper. Add the prepared Alcian Blue stain to the sections and incubate for 30 min at 37 &#176;C in a humidified chamber.</li>
		<li>Wash the unbound dye with 0.1 N HCl and then distilled water for 3 min. Dehydrate the sections in absolute ethanol, clear them in xylene, and finally mount the stained sections in a resinous medium for imaging.</li>
		<li>For Safranin-O staining, wash the hydrogel sections with distilled water 1x for 5 min. To fix the sections, add 4% NBF to the slides and incubate for 30 min. Wash the sections 1x with distilled water for 5 min, and then dip the slides in 1% acetic acid for 10-15 s. Dry the slides gently with tissue paper.</li>
		<li>Add prepared Safranin-O dye to the sections and incubate for 10 min at RT. Wash the unbound dye with 100% ethanol. Dip the slides in 100% ethanol for 5 min. Air-dry the slides, clear them in xylene, and finally mount the stained sections in a resinous medium for imaging.</li>
	</ol>
	</li>
	<li>Inducing osteogenic differentiation of IFP MSCs
	<ol>
		<li>To induce osteogenic differentiation, trypsinize the cells, as stated before (Step 2.2.). Seed the cells at a density of 3 x 103 cells/cm2 in a 24-well tissue culture plate. Add media to obtain a final volume of 500 &#181;L. Culture the cells at 37 &#176;C in a 5% CO2 incubator. 1 day post-seeding, replace the expansion media with 500 &#181;L osteogenic differentiation media.</li>
		<li>Prepare the osteogenic media by supplementing expansion media (DMEM + 10% FBS +2.5 &#181;g/mL amphotericin, 100 U/mL penicillin-streptomycin, and 25 &#181;g/mL ciprofloxacin) with 25 mM D (+)-glucose, 10 nM dexamethasone, 10 mM &#946;-glycerophosphate, 50 &#181;g/mL 2-Phospho-L-ascorbic acid trisodium salt, and 10 nM vitamin D3. 
		NOTE: The cells cultured in expansion media supplemented with 25 mM D (+) glucose are considered uninduced controls.</li>
		<li>Change the osteogenic media every 3 days for 14 days or 28 days. At the end of 14 days, measure the extent of osteogenesis by alkaline phosphatase (ALP) staining. 
		NOTE: To prepare the substrate for ALP staining, dissolve one 5-Bromo-4- Chloro-3-Indolyl Phosphate (BCIP)/Nitroblue Tetrazolium (NBT) tablet in 10 mL of distilled water in a 15 mL centrifuge tube. Let the tablet dissolve completely. The substrate solution is ready to use. Since the substrate solution cannot be stored for a longer duration, based on need, break the tablet in half and dissolve it in 5 mL of distilled water. To prepare the permeabilization buffer, add Tris buffer (100 mM) and magnesium chloride (5 mM) to distilled water. Add Triton X100 (0.1%) to the solution and let it dissolve. Adjust the pH of the solution to 9.25-9.75.</li>
		<li>For ALP staining, after 14 days of induction, remove the media and wash the cells with PBS. Fix the cells using NBF (4%) for 1 min. Wash the cells with PBS and permeate using the prepared lysis buffer for 2-3 min.</li>
		<li>Add 500 &#181;L of the prepared substrate solution to the cells and let it incubate for 15 min to 1 h, depending upon the expression level. Check the development of purple/blue color in between.</li>
		<li>Remove the substrate solution and wash with distilled water. Image the stained cells using an inverted microscope.</li>
		<li>At the end of 28 days, measure the extent of calcification after osteogenic induction by Alizarin Red-S staining. 
		NOTE: To prepare the Alizarin Red-S staining solution (40 mM), dissolve 2 g of Alizarin Red-S in 100 mL of distilled water and adjust the pH to 4.1-4.3 with HCl or NH4OH. Filter the solution through a 0.22 &#181;m membrane and store at 4 &#176;C in the dark. The pH of the solution is important; hence it is recommended either to make a fresh solution or to re-measure the pH of the solution if it is more than 1 month old.</li>
		<li>For Alizarin Red-S staining, after 28 days of induction, remove the media and wash the cells 1x with cold PBS. Fix the cells using 70% ethanol for 1 h at 4 &#176;C.</li>
		<li>Remove the fixative and wash the cells 2x with distilled water. Remove the water completely and add 1 mL of Alizarin Red-S solution to each well.</li>
		<li>Incubate the cells with the solution for 1 h at RT and image the cells using an inverted microscope.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no conflict of interest.

In the present protocol, a simple, reliable, and reproducible method for the isolation of MSCs from goat IFP has been provided. Cells isolated using this method have been successfully used in our previous studies for in vitro tissue regeneration. It was observed that the isolated cells were proliferative, were responsive to various growth factors, and retained their biological activity when seeded on electrospun fibers and scaffolds25,26. Moreover, it wa...

Mesenchymal stem cells (MSCs) are an attractive candidate for cell-based therapies in regenerative medicine1,2. They can be harvested from a variety of tissue sources such as bone marrow, umbilical cord, placenta, dental pulp, and subcutaneous adipose tissue3. However, as the availability of stem cells in adults is limited and their isolation procedure is often invasive (resulting in donor site morbidity), it is desirable to have an alternative stem cell source that could circumvent these challenges.
The knee joint is a depot of various cell types, such as infrapatellar fat pad-derived MSCs, synovial membrane-derived MSCs, synovial fluid-derived MSCs, ligament fibroblasts, articular chondrocytes, etc4,5,6. These cells have the potential to be widely explored in musculoskeletal tissue engineering-based research. Therefore, the knee joint could be a possible and reliable source of multiple types of MSCs. Adipose depot located in the knee joint, known as the infrapatellar fat pad (IFP) or Hoffa&#39;s fat pad, is a promising and alternative choice of MSC depot. The IFP is a relatively easily accessible and clinically obtainable source of MSCs, as it is routinely resected and discarded as surgical waste during knee arthroscopy or open knee surgery. Removal of the IFP is associated with minimal donor-site morbidity, which also makes it an attractive tissue source. While having a similar phenotypic profile, MSCs from IFP (IFP-MSCs) have enhanced clonogenic potential when compared with bone marrow-derived mesenchymal stem cells (BM-MSCs)6 and better proliferative capacity compared to subcutaneous adipose-derived stem cells (ADSCs)7. Interestingly, compared to synovial fluid-derived MSCs (SF-MSCs), IFP-MSCs do not lose their proliferative capacity at late passages, nor does doubling time increase at late passages. This suggests that, during cell expansion, IFP-MSCs can achieve a sufficiently large number of cells for in vitro tissue engineering applications without compromising their proliferation rate8. Recent studies have also suggested that IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ADSCs), probably due to their anatomical proximity to articular cartilage, indicating their suitability for cartilage tissue engineering6,7,9,10. Moreover, they also possess age-independent osteogenic differentiation potential11. Intra-articular injection of IFP-MSCs has been shown to reduce pain and improve knee joint functions in patients with osteoarthritis (OA)12,13. Further, strong immunosuppressive responses and improved immunomodulatory properties of IFP-MSCs in the presence of inflammatory cytokines during pathological conditions have also been reported6.
IFP-MSCs are a promising and alternate source of MSCs; however, their therapeutic benefit in tissue engineering and regenerative medicine is relatively less explored. The existing studies on IFP-MSCs have majorly utilized cells from human donors. Amongst these, a few recent studies have investigated IFP-MSCs from healthy human donors (non-arthritic patients, aged 17-60 years)6,14, whereas most of the studies have used IFP-MSCs from aged patients undergoing total knee replacement surgery (diseased patients, age 70-80 years). As both age and disease are known to alter the normal functioning of stem cells (reduced number and loss of functional potential), this could potentially lead to inconsistencies in the outcome of the MSC-based studies7,15,16,17. In addition to that, the use of IFP-MSCs from patients with pathophysiological conditions (e.g., arthritis and obesity) also poses difficulty for understanding the basic characteristics of healthy cells in vitro, thereby acting as a limiting factor in the development of MSCs-based therapies. To overcome these issues, the use of IFP-MSCs from healthy donors is vital. As access to a healthy human donor is challenging, animal models could be a better alternative. In this regard, there are a few studies where IFP has been isolated from mice18. However, owing to the small size of the fat pad in normal mice, fat tissues from multiple animals have been combined to get enough tissue to execute elaborate experimental procedures19. Hence, there is a need for a large animal model, which could fulfill the requirement for the higher number of cells and simultaneously comply with the 3R principles (refine, replace, and reduce) in animal research20. The usage of large animals has significant implications in translational research. Specifically, in musculoskeletal tissue engineering, a range of large animals such as dogs, pigs, sheep, goats, and horses have been investigated21. Goat (Capra aegagrus hircus) is an excellent choice of large animal since its stifle joint has the closest anatomy to the human knee joint22,23,24. The subchondral bone trabecular structure and subchondral bone thickness of goats are similar to humans, and the proportion of the cartilage to bone is also reported to be close to humans21. In addition, goats have been widely domesticated throughout the world, making them easily available when they are skeletally mature. Further, low maintenance costs and easy handling have made them an attractive animal model for research22.
In the present study, a simple protocol for the isolation of IFP-MSCs from the stifle joint of Capra aegagrus hircus (goat) and in vitro culture conditions for their expansion and differentiation are demonstrated. The isolated cells are adherent, have MSC-like morphology, form CFU-F (colony-forming unit-fibroblast) colonies, and possess adipogenic, chondrogenic, and osteogenic differentiation potential. Therefore, IFP-MSCs show potential as an alternative source of MSCs for biomedical applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-10G</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>0.25% Trypsin- 0.02% EDTA</td>
			<td>Hi-Media</td>
			<td>TCL049</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL centrifuge tube</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Phospho-L-ascorbic acid trisodium salt</td>
			<td>Sigma</td>
			<td>49752-10G</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>HiMedia</td>
			<td>TCL021-50ml</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>50-mL centrifuge tube</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Hi-Media</td>
			<td>RM471</td>
			<td>For sufated gycosaminoglycans staining</td>
		</tr>
		<tr>
			<td>Alizarin Red S</td>
			<td>S D Fine-Chem Limited</td>
			<td>26048-25G</td>
			<td>For calcium deposition</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>HiMedia</td>
			<td>A011</td>
			<td>2.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)</td>
			<td>Sino Biologicals</td>
			<td>10014-HNAE</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>BCIP/NBT ALP Substrate</td>
			<td>Sigma</td>
			<td>B5655-5TAB</td>
			<td>For ALP staining</td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>HiMedia</td>
			<td>MB-083</td>
			<td>Long name: Bovine Serum Albumin (1.25 mg/mL )</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>HiMedia</td>
			<td>TCP-182</td>
			<td>70 &#181;m</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>REMI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>RANBAXY LAB. Limited</td>
			<td>B17407T1</td>
			<td>2.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>S D Fine-Chem Limited</td>
			<td>42555</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-glucose</td>
			<td>Merck</td>
			<td>1.94925.0521</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D2915</td>
			<td>1 &#181;M</td>
		</tr>
		<tr>
			<td>DMEM LG</td>
			<td>SIGMA</td>
			<td>D5523</td>
			<td>Long name: Dulbecco&#8217;s Modified Eagle&#8217;s Media Low Glucose</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>Long name: Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Formaldehyde solution 37%-41%</td>
			<td>Merck</td>
			<td>61780805001730</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td>100 &#181;M</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td>10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon Eclipse TS 100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ITS + 1</td>
			<td>Sigma-Aldrich</td>
			<td>I2521-5mL</td>
			<td>Long name: insulin, transferrin, sodium selenite + linoleic-BSA</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>HiMedia</td>
			<td>TO-109-25G</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Merck</td>
			<td>61751605001730</td>
			<td>For lysis buffer</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Meck</td>
			<td>1.07018.2521</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes and sterile tips (20 &#181;L, 200 &#181;L, 1000 &#181;L)</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MUSE Cell analyser</td>
			<td>Merck Millipore</td>
			<td></td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Long name: Optimal Cutting Temperature</td>
		</tr>
		<tr>
			<td>Oil Red O dye</td>
			<td>S D Fine-Chem Limited</td>
			<td>54304</td>
			<td>For lipid vacuole staining</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>HiMedia</td>
			<td>A007</td>
			<td>100 U/mL</td>
		</tr>
		<tr>
			<td>Petri dishes (150 mm and 90 mm)</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>S D Fine-Chem Limited</td>
			<td>50240</td>
			<td>For sufated gycosaminoglycans staining</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3434</td>
			<td>3.4 % (w/v)</td>
		</tr>
		<tr>
			<td>Sterile scissors, forceps and scalpels</td>
			<td></td>
			<td></td>
			<td>For isolation of IFP-MSC</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>1.94953.0521</td>
			<td>35 % (w/v)</td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>Sino Biologicals</td>
			<td></td>
			<td>Long name: Transforming growth factor- &#946;1 (10 ng/mL)</td>
		</tr>
		<tr>
			<td>Tissue culture incubator 37 &#176;C, 5% CO2</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer</td>
			<td>Merck</td>
			<td>61771405001730</td>
			<td>For lysis buffer</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>S D Fine-Chem Limited</td>
			<td>40632</td>
			<td>For lysis buffer</td>
		</tr>
		<tr>
			<td>Type II collagenase</td>
			<td>Gibco</td>
			<td>17101015</td>
			<td>1.5 mg/mL</td>
		</tr>
		<tr>
			<td>Vitamin D3</td>
			<td>Sigma</td>
			<td>C9756-1G</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Well plates (6 -WP and 24-WP)</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, expansion, and differentiation of mesenchymal stem cells from the infrapatellar fat pad of the goat stifle joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Isolation of IFP-MSCs from the femorotibial joint of goat 
The steps involved in the isolation of IFP-MSCs from the stifle joint of a goat are depicted in Figure 1. The fat pad present in the inner non-articulating surface of the patella was removed, minced, and enzymatically digested. The IFP-MSCs were successfully isolated and cultured in vitro (Figure 2A).
Expansion and clonogenic ability of I...

Watch this Scientific Journal Video about Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint at JoVE.com

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

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1.7K Views.  Indian Institute of Technology-Kanpur. Infrapatellar fat pad mesenchymal stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

Video: Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.