3D Human Heart Microtissues

This microtissue was formed from a mechanically disaggregated sample taken during the implantation of a lower ventricular assist device.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates (see below) at 90% background oxygen partial pressure.  The samples were used during the COVID-19 Pandemic to study SARS-CoV-2 infection and interactions with cardiac tissues.  Viability was confirmed out to 45 days, although sample integrity was greatly diminished after 28 days.  These heart microtissue samples were roughly 400 um in diameter (largest dimension), and some began coordinated contractions after 3 days in culture.

This heart microtissue sample was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 7 days and stained for actin (TRITC: red), the nuclei (DAPI: blue), and for Troponin (FITC: green). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R. 

3D Colorectal Organoids

This colorectal organoid was formed from a mechanically disaggregated sample of the large intestine.  The microtissue fragment was initially faceted as a result of the mechanical disaggregation, but became smooth and spherical during a maturation process under perfusion in the 3D Darcy Plates.  At the time of imaging the sample was 14 days old, and had been under continuous perfusion in our Darcy plates at 90% background oxygen partial pressure.  This sample was used during an exploratory study of liquid culture medium tuning and optimization.  Viability was confirmed out to 21 days, at which time the experiments were stopped; the samples were still functional and viable.  The intestinal crypts formed on these organoids, which can be seen as the circular protuberances on the periphery.   These colorectal organoid samples were roughly 200-400 um in diameter.  The colorectal organoids produced copious amounts of mucin, and some showed prolonged coordinated contractions over weeks in 3D culture.

This colorectal organoid was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 14 days and stained for actin (TRITC: red), the nuclei (DAPI: blue), and E-cadherin (Cy5: white). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Glioma Tumoroids

This glioma cancerous “tumoroid” was formed from a mechanically disaggregated sample taken during a biopsy of the glioma.  The microtissue fragment was initially faceted as a result of the mechanical disaggregation with large amounts of necrotic tissue. These spherical tumoroids grew out of the original microtissue fragments over 3 days under perfusion in the 3D Darcy Plates.  At the time of imaging this sample was 14 days old, and had been under continuous perfusion with Neurocult liquid media with supplements and 5% fetal bovine serum in our Darcy plates at 90% background oxygen partial pressure. This sample was used as part of our in vitro experiments in immuno-oncology.  The tumoroids all grew to approximately 250 um in diameter, and all displayed similar sphericity and intact morphology.

This gioma tumoroid is stained with a viability stain Calcein-AM (FITC: green), a nuclear stain Hoechst- 33342 (DAPI: blue), and a dead stain BOBO-3 Iodide (TRITC: red).  Small necrotic fragments were found throughout the 3D medium from the beginning, and is characteristic of gliomas. 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

activated human immune cells in 3D

This collection of immune cells have been expanding over 2 weeks under perfusion in the 3D Darcy Plates.  At the time of imaging this aggregated collection of immune cells had been in culture for 8 days under continuous perfusion with RPMI 1640 with 10% human serum and 1% Glutamax with 60U/ml IL2.  Background oxygen partial pressures were approximately 20%.  These cells are used as part of our in vitro experiments in immuno-oncology, and are taken from collections of donor blood. Activation occurs in the Liquid Like Solid (LLS) 3D Culture medium using Dynabeads coated with aCD3 and aCD28 antibodies.  The beads are stationary within the LLS medium, and the immune cells move through interstitial space in the LLS, interact with the beads, proliferate, and expand in regional aggregates.

This immune cell aggregate was fixed and stained in situ in the LLS.  F-Actin is stained with AlexaFluor 555 (TRITC:red), and the nuclei were stained with NucBlue (DAPI: blue) 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D In vitro Immuno oncology experiment
immune cell killing of cancer

This image is taken from an experiment where donor matched immune cells and cancerous tumoroids were reintroduced in the 3D Liquid Like Solid (LLS) culture medium.  The movie is created from a series of images take over 16 hours.  The patient immune cells have been activated and treated with a checkpoint inhibitor prior to reintroduction with the tumoroid, which has been growing under continuous perfusion in our Darcy plates at approximately 20% background pressure of oxygen.  The immune cells move through interstitial space in the LLS, and had strong interactions with the tumoroid, which rapidly displayed detached nearly circular cell morphology and blebbing on the surface of the tumoroids indicative of immune cell mediated cancer cell killing.

This tumoroid is stained with a cell tracker stain CMFDA (FITC: green) and the immune cells are stained with cell tracker orange CMRA (TRITC: red)

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

movie: immune cells killing tumoroid

3D In vitro Immuno oncology experiment
Allogeneic immune cell killing of cancer

This image is taken from an experiment where donor mis-matched immune cells and cancerous tumoroids were introduced in the 3D Liquid Like Solid (LLS) culture medium.  The movie is created from a series of images take over 16 hours.  The patient immune cells have been activated prior to introduction with the tumoroid, which has been growing under continuous perfusion in our Darcy plates at approximately 20% background pressure of oxygen.  The immune cells move through interstitial space in the LLS, and had strong interactions with the tumoroid, which rapidly displayed detached nearly circular cell morphology and blebbing on the surface of the tumoroids indicative of immune cell mediated cancer cell killing.

This tumoroid is stained with a cell tracker stain CMFDA (FITC: green) and the immune cells are stained with cell tracker orange CMRA (TRITC: red)

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

movie: immune cells killing cancer

intestinal epithelial cell  (iec-6) organoids

This image is taken from an experiment 3D printing (see: “Writing in the Granular Gel Medium) IEC-6 cells (cell-line) and matured over 7 days under continuous perfusion in our Darcy plates at 20% background oxygen partial pressure.  This sample was used during an exploratory study of organoid biofabrication.  These intestinal organoids grew to roughly 200-400 um in diameter.  These organoids were extremely durable, and showed viability out to over 60 days, even in the absence of perfusion.  The cells showed strong motility along the surface of the organoid, which could be followed via in situ scanning confocal microscopy in the LLS medium.

This organoid was stained with for tight junction protein-1 (ZO-1) (TRITC:red), actin (FITC:green), and the nuclei were stained with NucBlue (DAPI:blue)

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Colorectal Organoids

This image is taken from a colorectal organoid that was formed from a mechanically disaggregated sample of the large intestine.  The microtissue fragment was initially faceted as a result of the mechanical disaggregation, but became smooth and spherical during a maturation process under perfusion in the 3D Darcy Plates.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates at 90% background oxygen partial pressure.  

This image is a confocal scanned cross-section of the surface in an effort to visualize the intestinal crypts.  This colorectal organoid was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 7 days and stained for F-actin using Phalloidin (FITC: red), the nuclei Hoechst 33342 (DAPI: blue), and E-cadherin (Cy5: shown red). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Colorectal microtissue explants and 3D Organoids

This colorectal organoid was formed from a mechanically disaggregated sample of the large intestine.  The microtissue fragment retained its initially faceted morphology from mechanical disaggregation during a maturation process under perfusion in the 3D Darcy Plates.  At the time of imaging the sample was 14 days old, and had been under continuous perfusion in our Darcy plates at 90% background oxygen partial pressure.  This sample was used during an exploratory study of liquid culture medium tuning and optimization.  Viability was confirmed out to 21 days, at which time the experiments were stopped; the samples were still functional and viable.  The intestinal crypts were retained on these organoids, which can be seen as the circular protuberances on the face of the microtissue explants.   These colorectal organoid samples were roughly 300-500 um in largest dimension.  These colorectal organoids and microtissue explants produced copious amounts of mucin, and some showed prolonged coordinated contractions over weeks in 3D culture.

This colorectal organoid was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 14 days and stained for for tight junction protein-1 (ZO-1) (FITC:green), actin Phalloidin (TRITC:red), the nuclei were stained with the nuclei Hoechst 33342 (DAPI: blue), and E-cadherin (Cy5: white). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Glioma Tumoroids

This pair of glioma cancerous “tumoroids” were formed from a mechanically disaggregated sample taken during a biopsy of the glioma.  The microtissue fragment was initially faceted as a result of the mechanical disaggregation with large amounts of necrotic tissue. These two spherical tumoroids grew out of the original microtissue fragments over 21 days under perfusion in the 3D Darcy Plates.  Continuous perfusion with Neurocult liquid media with supplements and 5% fetal bovine serum in our Darcy plates at 90% background oxygen partial pressure. These sample was used as part of our in vitro experiments in immuno-oncology.  The tumoroids all grew to approximately 250 um in diameter, and all displayed similar sphericity and intact morphology.

This gioma tumoroid is stained with a viability stain Calcein-AM (FITC: green), a nuclear stain Hoechst- 33342 (DAPI: blue), and a dead stain BOBO-3 Iodide (TRITC: red).  Note the necrotic fragment in the vicinity of the tumoroids, similar fragments were found throughout the 3D medium from the beginning, and are characteristic of gliomas. 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D In vitro Immuno oncology experiment
Immune cell tracking and T cell mediated killing of cancer

This image is taken from an experiment where donor mis-matched immune cells and cancerous glioblastoma tumoroids were introduced in the 3D Liquid Like Solid (LLS) culture medium.  The movie is created from a series of images take over 16 hours.  The patient immune cells have been activated prior to introduction with the tumoroid, which has been growing under continuous perfusion in our Darcy plates at approximately 20% background pressure of oxygen.  The immune cells move through interstitial space in the LLS, and had strong interactions with the tumoroid, which rapidly displayed contractions and detached nearly circular cell morphology and blebbing on the surface of the tumoroid.  Immune cells were sorted and stained prior to introduction into 3 groups: CD4+, CD8+, and other leukocytes.

This tumoroid is stained with a cell tracker stain CMFDA (FITC: green) and the immune cells are stained with cell trackers: CD8+ (TRITC:red), CD4+ (DAPI:cyan), other leukocytes (Cy5,white). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

movie: tracking immune cell killing

3D Human Lung Microtissue Model for COVID-19 and sars-cov-2 experiments

This microtissue is from a mechanically disaggregated sample taken from a non-cancerous region of lung after a lobectomy.  The aveolar space and structure were maintained as well as cellular heterogeneity and resident immune cells.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates (see below) at 90% background oxygen partial pressure.  The samples were used during the COVID-19 Pandemic to study SARS-CoV-2 infection and interactions with pulmonary tissues.  Viability was confirmed out to 14 days, although most infection and drug study experiments took only 72 hours. These lung microtissue samples were roughly 400 um in largest dimension.

This lung microtissue sample was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 7 days and stained for F-actin Phalloidin (FITC: green) and the nuclei were stained with Hoechst 33342 (DAPI: blue). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R. 

3D Human Lung Microtissue Model for COVID-19 and Coronavirus experiments

This microtissue is from a mechanically disaggregated sample taken from a non-cancerous region of lung after a lobectomy.  The aveolar space and structure were maintained as well as cellular heterogeneity and resident immune cells.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates (see below) at 90% background oxygen partial pressure.  The samples were used during the COVID-19 Pandemic to study SARS-CoV-2 infection and interactions with pulmonary tissues.  Viability was confirmed out to 14 days, although most infection and drug study experiments took only 72 hours. These lung microtissue samples were roughly 400 um in largest dimension.  The infection here, shown in TRITIC:red is from a human coronavirus OC43 (HCoV-OC43), and was performed in our BSL2 facility.

This lung microtissue sample was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 7 days and stained for F-actin Phalloidin (FITC: green) and the nuclei were stained with Hoechst 33342 (DAPI: blue), and HCoV-OC43 was stained with TRITC:red.

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Human Lung Microtissue Model for COVID-19 and sars-cov-2 experiments

This microtissue is from a mechanically disaggregated sample taken from a non-cancerous region of lung after a lobectomy.  The aveolar space and structure were maintained as well as cellular heterogeneity and resident immune cells.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates (see below) at 90% background oxygen partial pressure.  The samples were used during the COVID-19 Pandemic to study SARS-CoV-2 infection and interactions with pulmonary tissues.  Viability was confirmed out to 14 days, although most infection and drug study experiments took only 72 hours. These lung microtissue samples were roughly 400 um in largest dimension.  The infection here is with SARS-CoV-2, which was performed in a BSL3 facility.  

This lung microtissue sample was fixed in place and stained for F-actin Phalloidin (TITC: red). the nuclei were stained with Hoechst 33342 (DAPI: blue), and SARS-CoV-2 was stained with FITC:green.

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

DARCY plates

3D perfusion plates for long term culture of organoids, microtissue explants, and in vitro immuno ocology experiments.

This image is from a time lapse movie showing perfusion of the liquid media through the Liquid Like Solid (LLS) 3D culture medium.  The Darcy Plates use a passive negative pressure (sub ambient pressure) device to draw the liquid fluid through the LLS and around the tissue and cells in the LLS and into 4 isolated collection wells.  The plate has 24 individual experiments (24-wells) group in sets of 6 (4 quadrants) and allow for continuous and steady perfusion.  The effluent can be collected through a collection port without opening the Darcy place.  The gives passage free continuous culture of 3D tissues, organoids, and experiments with immune cells and drug screening.  The Darcy plates enable our immuno-oncology experiments and long duration 3D culture.  Although the Darcy plates are smaller than traditional 96-well plates, they work within standard cell culture infrastructure. 

movie: Darcy Plates 3D Perfusion

3D Printed osteosarcoma tumoroid

This patient derived osteosarcoma tumoroid was 3d printed and matured under perfusion in the 3D Darcy Plates with a background oxygen partial pressure of ~20%.  At the time of imaging this sample was 14 days old.  This sample was used during our studies in osteosarcoma and pediatric cancer.  Mitotic figures and nuclear atypia can be seen through this tumoroid.  The tumoroids maintained oncogenically active cells as can be seen through an oncogenic reported (FITC:green).

This osteosarcoma tumoroid was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 14 days and stained for actin (TRITC: red) and the nuclei (DAPI: blue). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

IN situ measurements of cytokine production

The Liquid Like Solid (LLS) 3D Culture medium facilitates the positioning of solid objects, cells, and tissues.  An osteosarcoma tumoroid (similar to above) is placed in the LLS with a field of ELISA beads that can be used to capture secreted cytokines.  Osteosarcoma is well known to produce Interleukin 8 (IL8), which is a chemokine.  Through a calibration procedure the fluorescence of the beads can be converted into a local concentration of IL8.  The movie shows the spatiotemporal measurements of IL8 around the osteosarcoma tumoroid.  These data can be fit to known solutions based on diffusion to solve for the surface concentration of IL8 at the tumor margin, as well as production rates of IL8 by the tumor.  These experiments are not performed under perfusion; the LLS suppresses convention, and in the absence of perfusion demonstrates nearly ideal Fickian diffusion behavior.

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

movie: Cytokine Production by Osteosarcoma

3D Pancreatic islet culture
experiments for Type-1 diabetes 

Human pancreatic tissue slices with islets were culture in 3D for 10 days under perfusion in the 3D Darcy Plates at 90% background oxygen partial pressure. The media was BrainPhys with supplements.  At the time of imaging the sample was 7 days old, although viability was confirmed out to 10 days.  In order to demonstrate functionality, the pancreatic tissues were removed from LLS culture and exposed to glucose challenges while measuring the production of glucagon and insulin.  

The image to the right is a high magnification images of an islet within the pancreatic microtissue.  Glucagon is indicated through the magenta stain and insulin is indicated through FITC:green.

3D Printed Renal Cell Carcinoma tumoroid

This renal cell carcinoma tumoroid was 3d printed in Liquid Like Solid (LLS) 3D medium and matured under perfusion in the 3D Darcy Plates with a background oxygen partial pressure of ~20%.  At the time of imaging this sample was 14 days old.  This sample was used during our studies in immuno oncology and the role of cancer associated fibroblasts.  These renal cell carcinoma tumoroids produce abundant extracellular matrix, which can be imaged and appears as a diffuse halo or corona around the tumoroids.  Cancer associated fibroblasts (CAFs) readily attached to this ECM and rapidly coated and overgrew the tumoroids.

This renal cell carcinoma tumoroid was fixed in place in the Liquid Like Solid (LLS) 3D culture medium after 14 days and stained for actin (TRITC:red), cell tracker CMFDA (FITC:green), and the nuclei were stained with the nuclei Hoechst 33342 (DAPI: blue). 

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

Hepatic Cancer micro-tumoroids

This liver cancer micro-tumoroid develops from single cells dispersed throughout the interstitial space of the Liquid Like Solid (LLS) 3D culture medium.  The incredibly large number of interstitial sites allow for 1×10⁶ (million) cell culture in a single well of approximately 200 ul of LLS.  Continuous perfusion in our Darcy plates, facilities continued growth and maturation over extended duration (weeks to months).  Isolating the cells from LLS can be done through a number of techniques from elutriation to cell screening and density based liquid separation techniques.  

This hepatic cancer tumoroid was isolated and stained with a viability stain Calcein-AM (FITC: green), a nuclear stain Hoechst- 33342 (DAPI: blue), and a dead stain BOBO-3 Iodide (TRITC: red).

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Human Colorectal Cancer TUmoroid

This human colorectal tumoroid was formed from a mechanically disaggregated sample taken from colorectal cancer surgical removal.  The microtissue fragment was initially faceted as a result of the mechanical disaggregation and has retained some of this form during a maturation process under perfusion in the 3D Darcy Plates.  At the time of imaging the sample was 14 days old, and had been under continuous perfusion in our Darcy plates at 90% background oxygen partial pressure.  This sample was used during our immuno oncology in vitro experiments.  Viability was confirmed out to 21 days, at which time the experiments were stopped; the samples were still functional and viable. These colorectal cancer tumoroid samples were roughly 100-400 um in diameter, and also produced copious amounts of mucin.

This colorectal cancer tumoroid was isolated and stained with a viability stain Calcein-AM (FITC: green) and a dead stain BOBO-3 Iodide (TRITC: red).

Image collected in the Cancer Engineering Laboratory on a fast scanning fluorescence confocal microscope, Nikon A1R.

3D Colorectal microtissue explants and 3D Organoids

This colorectal organoid was formed from a mechanically disaggregated sample of the large intestine.  The microtissue fragment retained its initially faceted morphology from mechanical disaggregation during a maturation process under perfusion in the 3D Darcy Plates.  At the time of imaging the sample was 7 days old, and had been under continuous perfusion in our Darcy plates at 90% background oxygen partial pressure.  This sample was used during an exploratory study of liquid culture medium tuning and optimization. These colorectal organoids and microtissue explants produced copious amounts of mucin, and some showed prolonged coordinated contractions over weeks in 3D culture.

This image is from a brightfield microscopy imaging timelapse movie over 10 minutes. showing the continuous and persistent contractions, which responded to drugs, calcium channel blockers, and epinephrine.  

movie: contractions of a gut microtissue

3D Printing in Liquid like solids
soft matter engineering

The whimsical jellyfish is being printed in a soft granular microgel bed of particles that we named Liquid-Like Solid (LLS).  We developed this model using free-form 3D biofabrication tools to support the precise printing of delicate structures in a completely scaffold-free self-healing 3D medium, LLS. Most of the images in this gallery were created in 3D LLS culture medium. These microgel particles settle into a particle bed with a low yield stress allowing the precise deposition and placement of cells and tissues; the LLS spontaneously resolidifies after placement. This enables the freeform deposition and arrangement of cells and biomaterials in any size, shape, or configuration (e.g. the jellyfish). These microgels are optically-matched with the liquid media and transparent, thereby facilitating high resolution in situ imaging and microscopy. The LLS microgel particles are charge-neutral, inert, and can be readily swollen in liquid media to support the growth and development of cells in 3D and facilitate non-destructive in situ imaging and quantification of fluorescent (or luminescent) tags and reporters to assess cell viability, motion, and pathway activation.

movie: 3D Printing in Liquid Like Solids