This research was supported by the National Health and Medical Research Council of Australia (#GNT1078247).

All animal procedures were conducted according to experimental protocols approved by the University of Queensland Animal Ethics Committee (UQBR/079/19).
1. Tissue collection and stromal cell preparation
<ol>
	<li>Euthanize 0.5-1.5 day-old male and/or female BALB/c neonatal donor mice by induction of hypothermia by wrapping animals inside the tissue paper and placing them under crushed ice for &#62;10 min. 
	NOTE: This protocol can be adapted to different mouse strains, ages, and the number of animals.</li>
	<li>Prepare sterile surgical instruments.</li>
	<li>Lay the mouse in a right lateral position. Swab the skin with 80% ethanol and make a 1 cm incision with Iris surgical scissors to expose the peritoneal wall.</li>
	<li>Make a second 0.5 cm incision above the spleen and use a pair of fine forceps to gently lift the tissue up. Use a pair of surgical scissors to cut the blood vessels and excise the tissue. Transfer the tissue into a Petri dish containing ice-cold Phosphate Buffered Saline (PBS). Remove any remaining connective tissue attached to the spleen. 
	NOTE: A stereomicroscope can assist fine motor movements required for manipulating surgical instruments.</li>
	<li>To dissociate whole tissues, transfer neonatal spleens (pools of 10 or less) into the inner rim of an inverted 14 mL conical tube cap. Mechanically disrupt tissues with a pressing motion using the plastic back end of a 1 mL syringe plunger.</li>
	<li>Place and secure the cap tightly over a 14 mL conical tube containing 10 mL of cold PBS. Invert the tube 5x to wash all disrupted tissue from the cap into the tube.</li>
	<li>Repeat step 1.6 for any remaining tissue.</li>
	<li>Leave the tube on ice for 1 min to let the tissue settle to the bottom of the tube.</li>
	<li>Carefully discard the supernatant (containing hematopoietic cells) by passing the solution through a reversible 70 &#181;m cell strainer.</li>
	<li>Recover the non-soluble stromal fraction by reversing the strainer and washing stromal tissue back into the 14 mL conical tube using freshly prepared 2 mL of supplemented Dulbecco&#39;s Modified Eagle Medium (sDMEM) containing 1 mg/mL Collagenase IV, 40 &#181;g/mL DNase I and 2% Fetal Bovine Serum (FBS). 
	CAUTION: Collagenase IV and Collagenase D are hazardous substances and must be handled inside a biosafety cabinet with appropriate personal protective equipment.</li>
	<li>To prepare a single cell suspension, enzymatically digest pooled tissue (up to 20) for 10 min at 37 &#176;C with constant rotation.</li>
	<li>Add 4 mL of freshly prepared sDMEM containing 1 mg/mL Collagenase D, 40 &#181;g/mL DNase I, and 2% FBS directly to the tube containing stromal tissue. Incubate for a further 10 min at 37 &#176;C with constant rotation.</li>
	<li>Halt the digestion by adding 8 mL of ice-cold PBS. Collect cells by centrifuging at 200 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant, and wash cells twice by resuspending in 1 mL of cold PBS and centrifuging at 200 x g for 5 min at 4 &#176;C. Keep cells on ice. 
	NOTE: Cells can be counted and adjusted to a desired concentration. This may require optimization for different cell populations. Using this protocol, 0.5 x 106- 1 x 106 stromal cells/spleen are typically recovered, depending on the age of donor mice. Cells can optionally be stained and FACS sorted at this stage to define cell composition.</li>
</ol>
2. Matrix encapsulation of cell aggregates
<ol>
	<li>Pre-chill sterile P200 pipette tips inside a -20 &#176;C freezer.</li>
	<li>Cut flexible laboratory film (e.g., Parafilm) into 1 cm x 2 cm pieces and sterilize by submerging in 80% ethanol for 10 min, followed by PBS for 10 min. Laboratory film can be prepared in advance and stored sterile.</li>
	<li>Prepare 0.5 x 106-2.5 x 106 cells by centrifuging at 200 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving approximately 20 &#181;L of the remaining PBS volume. Keep the cell pellet on ice. 
	NOTE: The remaining PBS can be gently aspirated without disturbing the cell pellet to confirm the volume.</li>
	<li>Aspirate 2 &#181;L of ice-cold basement membrane matrix into a pre-chilled P200 pipette tip, using a P20 pipettor. 
	NOTE: A highly concentrated basement membrane matrix (e.g., Corning Matrigel Matrix High Concentration) assists in forming a strong solidified plug.</li>
	<li>Gently twist to eject the pipette tip. Stretch a pre-sterilized strip of the laboratory film and place it over the end of the pipette tip, taking care not to pierce the film. Continue to wrap the tip in the film to seal the pipette tip. Carefully place the sealed tip directly on ice, taking care not to rupture the film.</li>
	<li>Resuspend the cell pellet in the remaining PBS and layer the solution gently over the basement membrane matrix. Keep the construct on ice.</li>
	<li>Prepare a centrifugation tube by placing a 1.2 mL cluster tube inside a 14 mL conical tube.</li>
	<li>Place the pipette tip inside the nested tube configuration and centrifuge at 400 x g and 4 &#176;C for 5 min.</li>
	<li>Position the 14 mL conical tube in a vertical orientation and incubate at 37 &#176;C for 15 min to allow the basement membrane matrix to solidify inside the pipette tip. 
	NOTE: The 14 mL conical tube can be placed on ice until required for downstream application.</li>
</ol>
3. Three-dimensional organoid culture
<ol>
	<li>Carefully remove the laboratory film from the pipette tip.</li>
	<li>Insert a thin stainless steel wire plunger through the larger opening of the pipette tip.</li>
	<li>Expel the matrix plug until released through the tip into one well of a non-treated 6-well tissue culture plate containing 2 mL of sDMEM supplemented with 10% FBS, 1x Glutamax, 1x Non-essential Amino Acids (NEAA), 10 &#181;M Rock Inhibitor, 10 U/mL Penicillin/Streptomycin, and 50 &#181;M &#946;-mercaptoethanol. 
	NOTE: The tissue culture vessel and corresponding media volume can be adjusted as required. 
	CAUTION: NEAA, Penicillin/Streptomycin, and &#946;-mercaptoethanol are hazardous substances and must be handled inside a biosafety cabinet with appropriate personal protective equipment.</li>
	<li>Culture organoids at 37 &#176;C, 5% CO2, and 95% humidity for 4-12 weeks.</li>
	<li>Replace half the medium every 5 days. 
	NOTE: If organoids begin attaching to the tissue culture plate, transfer to a fresh well.</li>
</ol>
4. Kidney capsule transplantation
<ol>
	<li>Prepare sterile surgical instruments.</li>
	<li>Perform a subcapsular kidney transplantation of the basement membrane matrix plug:
	<ol>
		<li>Anesthetize 8-week old BALB/c female recipient mouse with isoflurane following Institutional Animal Ethics Guidelines. 
		CAUTION: Isoflurane is a hazardous substance. Waste gas must be scavenged to prevent entry into the workspace environment.</li>
		<li>Lay the mouse in a right lateral position.</li>
		<li>Shave hair from the surgery site and disinfect the skin following Institutional Guidelines. 
		NOTE: It is recommended that the surgical site be disinfected three times in a circular motion with alternating iodine-based or chlorhexidine-based and alcohol-based scrub.</li>
		<li>Make a 2 cm incision in the skin perpendicular to the spine to expose the peritoneal wall. 
		NOTE: Fine surgical scissors or a scalpel blade can be used for skin incision. Users should follow Institutional Animal Ethics Guidelines or Standard Operating Procedures.</li>
		<li>Make a smaller 0.5 cm incision in the peritoneal wall above the kidney. 
		NOTE: The incision should approximate the kidney width to ensure that the kidney remains exteriorized during transplantation.</li>
		<li>Exteriorize the kidney through the peritoneal opening by applying downward pressure using the thumb and index finger. A pair of ring tweezers can be used to assist exteriorization. Ensure the kidney is moist throughout the procedure by regularly applying sterile PBS using a cotton swab.</li>
		<li>Under a stereomicroscope, pinch the perirenal fat of the kidney capsule with a pair of bent ultra-fine forceps at one pole of the kidney. Use a second pair of bent ultra-fine forceps to gently tear the kidney capsule membrane upwards in an opposing direction, creating a small opening.</li>
		<li>Carefully insert the prong of one forceps under the capsule membrane. Use a slow sweeping motion to separate the capsule membrane from the kidney parenchyma.</li>
		<li>Prepare the basement membrane matrix plug for transplantation by removing laboratory film from the pipette tip.</li>
		<li>Lift the kidney capsule using one prong of the forceps and insert the pipette tip through the opening, pushing it towards the opposing pole of the kidney.</li>
		<li>Insert a wire plunger into the pipette tip and expel the matrix plug whilst simultaneously withdrawing the pipette tip from the kidney.</li>
		<li>Moisten the kidney with PBS and re-internalize.</li>
		<li>Close the peritoneal wall with one 5-0 Vicryl suture and close the skin with two autoclips.</li>
		<li>Administer analgesic (Buprenorphine at 0.05-0.1 mg/kg subcutaneously or following Institutional Animal Ethics Guidelines). 
		CAUTION: Buprenorphine is a hazardous substance and must be handled with appropriate personal protective equipment.</li>
	</ol>
	</li>
	<li>Turn off the flow of isoflurane but keep the oxygen flow on to allow the mouse to breathe pure oxygen until it starts gaining consciousness.</li>
	<li>Return the mouse to its cage. Keep the animal warm during recovery by partially placing the cage on top of a heating pad or under a heat lamp, and monitor until the mouse fully recovers from anesthesia.</li>
	<li>Monitor post-surgical recovery for the first two weeks or as required by Institutional Guidelines.</li>
</ol>

<ol>
	<li>Jarcho, S., Andersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+autotransplantation+of+splenic+tissue.">Traumatic autotransplantation of splenic tissue.</a> American Journal of Pathology. 15 (5), 527-546 (1939).</li><li>Storsteen, K. A., ReMine, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rupture+of+the+spleen+with+splenic+implants:+splenosis.">Rupture of the spleen with splenic implants: splenosis.</a> Annals of Surgery. 137 (4), 551-557 (1953).</li><li>Mizrahi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraumatic+autotransplantation+of+spleen+tissue.">Posttraumatic autotransplantation of spleen tissue.</a> Archives of Surgery. 124 (7), 863 (1989).</li><li>Miko, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+autotransplantation.+Morphological+and+functional+follow-up+after+spleen+autotransplantation+in+mice:+A+research+summary.">Spleen autotransplantation. Morphological and functional follow-up after spleen autotransplantation in mice: A research summary.</a> Microsurgery. 27 (4), 312-316 (2007).</li><li>Grikscheit, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+spleen+protects+against+overwhelming+Pneumococcal+sepsis+in+a+rodent+model.">Tissue-engineered spleen protects against overwhelming Pneumococcal sepsis in a rodent model.</a> Journal of Surgical Research. 149 (2), 214-218 (2008).</li><li>Pabst, R., Westermann, J., Rothkotter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoarchitecture+of+regenerated+splenic+and+lymph+node+transplants.">Immunoarchitecture of regenerated splenic and lymph node transplants.</a> International Review of Cytology. 128, 215-260 (1991).</li><li>Tan, J. K. H., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+spleen+tissue+regeneration+from+neonatal+spleen+capsule+requires+lymphotoxin+priming+of+stromal+cells.">Murine spleen tissue regeneration from neonatal spleen capsule requires lymphotoxin priming of stromal cells.</a> The Journal of Immunology. 193 (3), 1194-1203 (2014).</li><li>Tan, J. K. H., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+subsets+directing+neonatal+spleen+regeneration.">Stromal cell subsets directing neonatal spleen regeneration.</a> Scientific Reports. 7 (1), 40401 (2017).</li><li>Gee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+organoid+units+generate+functional+human+and+mouse+tissue-engineered+spleen+in+a+murine+model.">Spleen organoid units generate functional human and mouse tissue-engineered spleen in a murine model.</a> Tissue Engineering Part A. 26 (7-8), 411-418 (2020).</li><li>Ueno, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Tlx1+marks+a+subset+of+lymphoid+tissue+organizer-like+mesenchymal+progenitor+cells+in+the+neonatal+spleen.">Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen.</a> Scientific Reports. 9 (1), 20408 (2019).</li><li>. Online data repository: Matrigel encapsulated cell aggregation for investigating murine spleen tissue formation Available from: <a target="_blank" href="https://osf.io/ehrbw/?view_only=7d6a8e05c84144d3a12d36ffe7f94f01">https://osf.io/ehrbw/?view_only=7d6a8e05c84144d3a12d36ffe7f94f01</a> (2023)</li></ol>

The authors have nothing to disclose.

The aggregation of neonatal spleen cells inside a semi-solid medium represents a viable method for generating spleen constructs. Similar basement membrane matrix-based protocols have been used to initiate three-dimensional spleen cultures10. Here, we demonstrate that spleen constructs are equally amenable to in vitro organoid culture systems as well as to in vivo transplantation models. Of note, the transplantation of in vitro cultured spleen organoids has not yet been t...

Traumatic rupture of the spleen and the appearance of multiple splenic nodules in the body was one of the first indications that spleen tissue harbored regenerative capacity1,2. Spleen auto-transplantations were later introduced into the clinic to preserve spleen tissue in patients requiring emergency splenectomy3. Yet, despite being a part of clinical practice for decades, very little is known about how the spleen regenerates. Animal transplantation models have provided insight into multiple parameters of spleen regeneration and immune function4,5. In particular, experimental modifications to the graft preparation method have allowed tissue regeneration to be studied in greater detail at the cellular and molecular level.
Transplantations involving whole spleen slices undergo a phase of mass necrosis before a new spleen structure is rebuilt6. The initial phase of graft necrosis suggests that the bulk of transplanted tissue largely consists of red and white blood cells and is unnecessary for spleen regeneration. This was investigated experimentally by excluding hematopoietic cells from spleen grafts before transplant under the mouse kidney capsule. Here, the non-leukocyte/non-erythrocyte fraction of the spleen, which includes stromal and endothelial cells, was shown to be sufficient to induce de novo tissue formation7. Spleen stromal tissue could be further processed into a single-cell suspension, enabling the use of cell sorting technologies to manipulate cellular graft composition. By selectively removing candidate cell types, two CD45-TER-119- stromal cell populations were identified that were indispensable for graft development: an endothelial-like CD31+CD105+MAdCAM-1+ cell population and a more broadly defined PDGFR&#946;+ mesenchymal cell population8.
The construction of grafts from spleen cells varies in terms of support materials and cell-loading processes. Tissue-engineered spleens have previously been prepared by loading splenic units onto a polyglycolic acid/poly-L lactic acid polymer scaffold5,9. Interestingly, spleen stromal cells absorbed into a collagen sponge failed to engraft, whereas stromal cells aggregated and loaded over a collagen sheet facilitated spleen regeneration8. The resuspension of spleen stromal cells inside a Matrigel matrix has also been demonstrated to induce cell aggregation under three-dimensional culture conditions10. However, this method has not been tested for use in transplantation models. The overall goal of the current protocol is to forcibly aggregate and encapsulate spleen stromal cells directly within the basement membrane matrix, which subsequently can be transferred to a three-dimensional in vitro tissue culture system or used as a vehicle for animal model transplantations (Supplementary Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well Polypropylene 1.2 mL Cluster Tubes</td>
			<td>Corning</td>
			<td>CLS4401</td>
			<td>For placing inside a 14 ml conical tube</td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Stock 55 mM, use at 50 uM</td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase I)</td>
			<td>Sigma-Aldrich</td>
			<td>D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas,Type II-S, lyophilized powder, Protein &#8805;80 %, &#8805;2,000 units/mg protein</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td>4500 mg/L Glucose, L-Glutamine, and Sodium Bicarbonate, without Sodium Pyruvate, Liquid. Sterile Filtered.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Without calcium chloride and magnesium chloride, sterile-filtered</td>
		</tr>
		<tr>
			<td>Eclipse 200 &#956;l Pipette Tips</td>
			<td>Labcon</td>
			<td>1030-260-000</td>
			<td>Bevel Point</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>Lot# 1382243</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Stock 100X, use at 1X</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354263</td>
			<td>Matrigel matrix basement membrane High Concentration, Lot# 7330186</td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>Stock 100X, use at 1X</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Stock 10,000 units/ml Penicillin, 10,000 ug/ml Streptomycin</td>
		</tr>
		<tr>
			<td>Reversible Cell Strainer</td>
			<td>STEMCELL Technologies</td>
			<td>27216</td>
			<td>70 &#956;m</td>
		</tr>
		<tr>
			<td>Ring Tweezers</td>
			<td>NAPOX</td>
			<td>A-26</td>
			<td>Ring size: 3 mm</td>
		</tr>
		<tr>
			<td>Rock Inhibitor (Y-27632)</td>
			<td>MedChemExpres</td>
			<td>HY-10071</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermofisher Heraeus Megafuge 40R Centrifuge</td>
			<td>Thermofisher</td>
			<td></td>
			<td>Acceleration and deceleration speeds were set to 8</td>
		</tr>
		<tr>
			<td>Ultra Fine Tweezers</td>
			<td>EMS</td>
			<td>78340-51S</td>
			<td>Style 51S. Antimagnetic/anti-acid SA low carbon austenitic steel tweezers are corrosion resistant. Anti-glare satin finish.</td>
		</tr>
		<tr>
			<td>Vicryl 5/0 Suture Ligapak Reel</td>
			<td>Ethicon</td>
			<td>J283G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wiretrol II Long Wire Plunger</td>
			<td>Drummond</td>
			<td>5-000-2002-L</td>
			<td>Stainless Steel Plunger, 25 &#38; 50 &#956;L/WRTL II, Long&#160;</td>
		</tr>
		<tr>
			<td>Wound Clip Applier</td>
			<td>MikRon</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound Clips</td>
			<td>MikRon</td>
			<td>427631</td>
			<td>9 mm</td>
		</tr>
	</tbody>
</table>

basement membrane matrix encapsulated cell aggregation for investigating murine spleen tissue formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

Cell aggregation is important for promoting cell-to-cell contact and signaling. Encasing cell aggregates inside the basement membrane matrix supported both 3-dimensional cultures for in vitro tissue organoid formation and facilitated the mechanical delivery of cells into the kidney capsule for graft transplantation. To establish these constructs, the basement membrane matrix was first maintained in a fluidic state under ice-cold conditions. Cell aggregation was subsequently achieved by layering a concentrated ce...

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

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Developmental Biology

394 Views.  Bond University. This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

Video: Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...