We thank Dr. Laura L. Bronsart for providing GFP and dTomato-labeled RAW 264.7 macrophages. This research was financially supported by NIH grant #R00CA201304.

Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparation of mice and cell acquisition (adapted from Nguyen-Ngoc et al.11)
<ol>
	<li>Sacrifice athymic Nu/Nu mice (8-10 weeks old) using CO&#173;2 asphyxiation followed by cervical dislocation. Clean the skin using 70% ethanol.</li>
	<li>Resect abdominal and inguinal mammary glands from mice using pre-sterilized scissors and forceps. Remove lymph nodes before resection. Rinse in sterile 1x phosphate buffered saline (PBS) (Figure 1A).</li>
	<li>Place it in 15 mL tubes with 10 mL of Dulbecco&#8217;s Modified Eagle Media/Nutrient Mixture F12 (DMEM/F12) for transport. Samples can be kept overnight at 4 &#176;C or processed immediately. Keep on ice.</li>
	<li>Irradiate samples at 20 Gy using a cesium source (Figure 1B).</li>
	<li>45 min after irradiation, place mammary glands in a 35 mm sterile cell plate and mince with scalpels (Figure 1C,D). Mince with approximately 40 strokes until the tissue relaxes and pieces are obtained that are no larger than approximately 1 mm2 in area.</li>
	<li>Transfer to the collagenase solution in a 50 mL centrifuge tube. Collagenase solution consists of 2 mg/mL collagenase (see Table of Materials), 2 mg/mL trypsin, 5% v/v fetal bovine serum (FBS), 5 &#956;g/mL insulin, and 50 &#956;g/mL gentamicin in DMEM/F12 media. Use 10 mL collagenase solution per mouse.</li>
	<li>Place in a water bath at 37 &#176;C, vortexing every 10 min for 30-60 min. Digestion is complete when the collagenase solution is cloudy (Figure 1E,F).</li>
	<li>Spin down the digested solution at 450 x g for 10 min at room temperature (RT). Three layers will be observed. The supernatant is composed of fat, the middle layer is an aqueous solution, and the bottom is a pellet. The pellet will appear red as it is a mixture of epithelial cells, individual stromal cells, and red blood cells (Figure 1G).</li>
	<li>Precoat all pipettes, pipette tips, and centrifuge tubes with bovine serum albumin (BSA) solution prior to contact. BSA solution consists of 2.5 w/v % BSA in Dulbecco&#8217;s Phosphate Buffered Saline (DPBS). For pre-coating, simply add then remove BSA solution to the inside of the pipette tip and tubes. BSA solution can be reused, although it should be sterile filtered before each experiment.</li>
	<li>For additional recovery, transfer the supernatant to a fresh BSA coated 15 mL tube. Pipette up and down vigorously to disperse fat layer. Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant, leaving a small amount of media in the tube to avoid aspirating the cell pellet.</li>
	<li>Aspirate the aqueous layer from the tube with the original pellet.</li>
	<li>Add 10 mL of DMEM/F12 to the tube with the original pellet and transfer to the second tube. Pipette vigorously to combine and resuspend the two pellets.</li>
	<li>Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant and add 4 mL of DMEM/F12 to the tube.</li>
	<li>Add 40 &#956;L of deoxyribonuclease (DNase) to the suspension and gently shake by hand for 2-5 min at RT. DNase solution consists of 4 U/mL DNase in DMEM/F12.</li>
	<li>Add 6 mL of DMEM/F12 and pipette thoroughly. Centrifuge the tube at 450 x g for 10 min at RT.</li>
	<li>Aspirate supernatant to the 0.5 mL mark. Resuspend in 10 mL of DMEM/F12 and pipette thoroughly.</li>
	<li>Pulse to 450 x g and stop 4 s after reaching that speed.</li>
	<li>Repeat steps 1.16-1.17 three more times to purify organoids via centrifugal differentiation. The pellet should now be an off-white color consisting of only epithelial organoids (Figure 1H). 
	NOTE: Organoids can also be filtered using sterile mesh 40 &#956;m filters. After step 1.16, pipette media containing organoids through a filter into a centrifuge tube, and then rinse with 5 to 10 mL of DMEM/F12 media. Flip the filter over a new 50 mL centrifuge tube. Pass 10 mL of DMEM/F12 media through, going the opposite way to rinse off any retentate. The retentate should consist of organoids, and the filtrate should consist mainly of stromal cells, which can be discarded or kept if desired.</li>
</ol>
2. Determining density and plating organoids
<ol>
	<li>Resuspend pellet in 10 mL of DMEM/F12. Pipette thoroughly to create a homogenous solution.</li>
	<li>Transfer 50 &#181;L to a 30 mm Petri dish, and view under a phase contrast microscope at 20x. Count the number of organoids with a tally counter. 
	NOTE: Here pipette tips have been consistently used with a minimal diameter of 457 &#956;m, which is 5-10 times the diameter of the organoids that are seeded. For transferring volumes of 2 mL or larger (e.g., steps 1.16 and 2.1), use serological pipettes with tip diameters excess of 1,500 &#956;m.</li>
	<li>Calculate the organoid density using the following equation: 
	<img alt="Equation" src="/files/ftp_upload/59293/59293eq1.jpg" /> 
	The desired density is 1,000 organoids/mL to simplify further dilution. If the density is too low, centrifuge at 450 x g for 5 min and aspirate media. Add media necessary to reach 1,000 organoids/mL, and pipette thoroughly to create a homogenous mixture.
	<ol>
		<li>To grow organoids in a protein matrix, seed organoids at a concentration of 1 organoid/&#956;L in collagen type 1 diluted to 87% or in basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma. While working with samples, keep on ice.</li>
		<li>To freeze organoids, transfer the desired volume to a separate centrifuge tube. Spin down at 450 x g for 5 min. Aspirate media, and then add the same volume of 90% FBS/10% DMSO. Resuspend the organoids, and then aliquot into cryotubes. Transfer to -80 &#730;C, and then to liquid nitrogen within one week.</li>
		<li>To thaw, warm in a 37 &#730;C water bath for one min. Centrifuge at 450 x g for 5 min, and then aspirate freezing media. Rinse with sterile DPBS, and then centrifuge again. Aspirate DPBS and add organoid media.</li>
	</ol>
	</li>
	<li>Pipette 50 &#181;L (50 organoids) into each well of the low adhesion plate (Figure 1I).</li>
	<li>Add 150 &#181;L of organoid media to bring the total working volume to 200 &#181;L. Organoid media consists of 1% penicillin-streptomycin and 1% insulin-transferrin-selenium (ITS) in DMEM/F12 media.</li>
	<li>Every 2 days replace media carefully. 
	NOTE: Low adhesion plates are not tissue culture treated; therefore, the cells can be easily detached. Aspirate media slowly by tilting the plate and inserting the pipette tip at the edge of each well. Leave a small amount of media in the bottom of the well. Add new media slowly to avoid applying unnecessary shear forces to organoids.</li>
</ol>
3. Co-culturing with macrophages
<ol>
	<li>Maintain GFP or dTomato-labelled RAW 264.7 macrophages in DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin. Seed 1 x 104, 5 x 104, or 1 x 105 cells/mL into organoid media.</li>
	<li>Use live cell phase contrast and fluorescent imaging to monitor macrophage infiltration over time.</li>
</ol>
4. Immunofluorescence staining of organoids
NOTE: Organoids can be stained in low adhesion wells or can be transferred to chamber slides. To transfer, gently pipette up and down until organoids have detached from plates. Transfer to chamber slides and incubate for 4-8 h to allow organoids to adhere to the plate surface.
<ol>
	<li>Remove organoid medium from the wells by carefully aspirating. Fix samples with 10% neutral buffered formalin for 15 min at RT.</li>
	<li>Wash 3x 5 min in 1x PBS. If desired, fixed samples can be stored at 4 &#176;C for one week for further staining.</li>
	<li>Permeabilize with 0.1% 4-(1,1,3,3-Tetramethylbutyl) phenyl-polyethylene glycol for 5 min. 
	NOTE: To stain for F-actin, incubate samples with phalloidin diluted 1:1,000 and 1.67 nM bisbenzimide nuclear dye in 1% PBS/BSA for 1 h at RT. Then, proceed to step 4.8.</li>
	<li>Bermeabilize with 0.5PBS. Samples can be stored at 4opy images and immunofluorescent images. mechanisms that contribute to CTC rlock with 5% normal goat serum in 0.1% PBS/Polyethylene glycol sorbitan monolaurate (PBST) for 1 h at RT. Wash 3x 5 min with PBS.</li>
	<li>Incubate with Anti-Cytokeratin 14 diluted 1:1,000, E-Cadherin diluted 1:200, or Tight Junction Protein One diluted 1:100 in 1% NGS in PBST for 1 h at RT. Wash 3x 5 min in PBST.</li>
	<li>Incubate with Goat Anti-Rabbit secondary diluted 1:200 with 1% NGS/PBST for 1 h at RT. Cover with foil to avoid light exposure.</li>
	<li>Wash 3x 5 min in PBS. Use the nuclear dye (see Table of Materials) to stain nuclei.</li>
	<li>Wash 3x 5 min in PBS. If using chamber slide, mount with a coverslip. Store wrapped in foil at 4 &#176;C for up to 2 weeks.</li>
</ol>

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The authors have nothing to disclose.

In this protocol, we have developed a method for reproducible growth and characterization of irradiated mammary organoids (Figure 1). An irradiation dose of 20 Gy was applied to mirror previous in vivo models of tumor cell recruitment5. Irradiation of mammary glands ex vivo prior to organoid formation allowed for isolation of radiation damage effects without a corresponding infiltration of immune cells. The development of an in vitro irradiated normal tissue model ena...

Approximately 60% of the triple negative breast cancer (TNBC) patients choose breast-conserving therapy (BCT) as a form of treatment1. In this treatment modality, the tumor containing part of the breast tissue is removed, and the surrounding normal tissue is exposed to ionizing radiation to kill any residual tumor cells. Treatment reduces recurrence in much of the breast cancer population; however, approximately 13.5% of treated patients with TNBC experience locoregional recurrences2. Therefore, studying how radiation may recruit circulating tumor cells (CTCs) will lead to important insights into local recurrence3,4.
Previous work has shown that radiation of the normal tissue increases recruitment of various cell types5. In pre-clinical models of TNBC, irradiation of normal tissue increased macrophage and subsequently tumor cell recruitment to normal tissues5. Immune status influenced tumor cell recruitment to irradiated sites, with tumor cell migration observed in immunocompromised subjects. Recapitulating these interactions using organoids derived from mammary glands will allow the observation of cell migration and cell-stromal interactions in real time with microscopy and live cell imaging to determine the role of radiation damage in altering tumor cell behavior.
Mouse mammary organoids have helped elucidate key steps in the development of the mammary gland. A mammary organoid is a multicellular, three dimensional construct of isolated mammary epithelium that is larger than 50 &#956;m6,7,8,9,10. Using primary epithelial organoids, Simian et al. evaluated necessary factors for branching in the mammary gland7. Shamir et al. discovered that dissemination can occur without an epithelial to mesenchymal transition, providing insight into the metastatic cascade8. Methods for generating and characterizing organoids from mammary gland tissue are well established6,11,12,13. However, to our knowledge, methods for growing irradiated organoids from mammary glands have not been reported. A protocol for growing and characterizing irradiated organoids would be a critical step in recapitulating radiation-induced immune and tumor cell recruitment.
In this paper, we report a method for growing and characterizing irradiated mammary epithelial organoids in low adhesion microplates coated with a hydrophilic polymer that supports the formation of spheroids. These organoids were co-cultured with macrophages to examine immune cell infiltration kinetics. This work can be extended to include co-culturing organoids with adipose cells to recapitulate mammary characteristics, breast cancer cells to visualize tumor cell recruitment, and CD8+ T cells to study tumor-immune cell interactions. Previously established protocols may be used to evaluate irradiated organoids. Earlier models co-culturing mammary organoids and immune cells have shed light on mechanisms of metastasis and dissemination. DeNardo et al. found that CD4+ T cell regulation of tumor associated macrophages enhanced a metastatic phenotype of mammary adenocarcinomas14. Co-culture models have also been used to elucidate mechanisms of biological development. Plaks et al. clarified the role of CD4+ T cells as down-regulators of mammary organogenesis15. However, our group is the first to establish a procedure of visualizing how normal tissue irradiation influences immune cell behavior. Because normal tissue irradiation has been shown to enhance tumor cell recruitment5, this protocol can be further developed to analyze how tumor cell behavior is altered by irradiation of normal tissue and cells, leading to a greater understanding of cancer recurrence.

<table>
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>16004-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cytokeratin 14</td>
			<td>abcam</td>
			<td>ab181595</td>
			<td>Lot: GR3200524-3</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium Histolyticum, Type VIII</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Fisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Cadherin</td>
			<td>Cell Signaling</td>
			<td>24E10</td>
			<td>Lot: 13</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td>green 
			Lot: GR3203000-1</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>abcam</td>
			<td>ab150080</td>
			<td>red 
			Lot: GR3192711-1</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Fisher</td>
			<td>62249</td>
			<td>Lot: TG2611041</td>
		</tr>
		<tr>
			<td>Insulin (10 mg/mL)</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium, 100x</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma)</td>
			<td>Corning</td>
			<td>356237</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclon Sphera 96 well plates</td>
			<td>Thermo</td>
			<td>174927</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>10128-856</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>abcam</td>
			<td>ab176757</td>
			<td>Lot: GR3214582-16</td>
		</tr>
		<tr>
			<td>Tight Junction Protein 1</td>
			<td>Novus</td>
			<td>NBP1-85047</td>
			<td>Lot: C115428</td>
		</tr>
		<tr>
			<td>Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (Polyethylene glycol sorbitan monolaurate)</td>
			<td>Sigma</td>
			<td>P1379-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

growth and characterization of irradiated organoids from mammary glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Irradiated epithelial mammary organoids were successfully obtained from mouse mammary glands, processed, and cultured on low-adhesion plates (Figure 1). Organoid yield was tested by seeding in different growth environments (Figure 2A-G). Seeding cells directly onto tissue culture treated 10 cm cell plates yielded an overgrowth of fibroblast cells. Fibroblasts were identified under phase contrast microscopy in or near the same plane of focus as organoids, and they quickly gre...

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Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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Research

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Bioengineering

8.0K Views.  Vanderbilt University. Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Video: Growth and Characterization of Irradiated Organoids from Mammary Glands

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

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 ...