Type I collagen self-assembles into three-dimensional (3D) fibrous networks. These dynamic viscoelasticmaterials can be remodeled in response to mechanical and chemical signals to form anisotropicnetworks, the structure of which influences tissue development, homeostasis, and disease progression.Conventional approaches for fabricating anisotropic networks of type I collagen are often limitedto unidirectional fiber alignment over small areas. Here, we describe a new approach for engineeringcell-laden networks of aligned type I collagen fibers using 3D microextrusion printing of a collagenMatrigel ink. We demonstrate hierarchical control of 3D-printed collagen with the ability to spatiallypattern collagen fiber alignment and geometry. Our data suggest that collagen alignment results froma combination of molecular crowding in the ink and shear and extensional flows present during 3Dprinting. We demonstrate that human breast cancer cells cultured on 3D-printed collagen constructsorient along the direction of collagen fiber alignment. We also demonstrate the ability to simultaneouslybioprint epithelial cell clusters and control the alignment and geometry of collagen fibers surroundingcells in the bioink. The resulting cell-laden constructs consist of epithelial cell clusters fully embeddedin aligned networks of collagen fibers. Such 3D-printed constructs can be used for studies ofdevelopmental biology, tissue engineering, and regenerative medicine.
This system includes two bone marrow lines, Kasumi-1 myeloblasts and MEG-01 megakaryocytes, along with primary human hepatocytes to measure the effects of drugs on bone marrow proliferation and on liver with organ-organ interactions between the liver and bone marrow.
Flow within the system is created by a rocking platform with a defined action of 1 oscillation per minute at an amplitude of 1 degree. This creates a recirculating flow between the system's various chambers.
McAleer et al. Sci Transl Med 2019
This system contains both an MDR+ vulva carcinoma–derived cell line (SW-962) and an MDR− breast cancer cell line (MCF-7), a liver compartment, and two separate bioMEMS devices to measure cardiac electrical and mechanical function.
Cardiac mechanical function is evaluated by incorporating the cardiomyocytes onto custom arrays of microscale cantilevers and calculating force and frequency dynamics from laser-based measurements of cantilever bending resulting from cardiomyocyte contractions.
Cardiac electrical function was measured via a MEA amplifier system by incorporating and chemically patterning cardiomyocytes onto cMEAs to produce a defined conduction path along a series of surface-embedded microelectrodes. Electrically stimulated cardiomyocyte activity was generated inside the system with housing-embedded electrodes for cantilevers and via stimulation through the MEA chip for electrical measurements.
Flow within the system is created by a rocking platform with a defined action of 1 oscillation per minute at an amplitude of 1 degree. This creates a recirculating flow between the system's various chambers.
McAleer et al. Sci Transl Med 2019
Bone was grown from adult hMSC capable of osteogenic differentiation within native bone ECM serving as a structural scaffold. An in-vitro model of Ewing’s sarcoma that mimics the key properties of the native tumor and provides the tissue context and physical regulatory signals.
A reproducible 3D neural constructs that incorporates vascular and microglial components derived by culturing precursor cells from the H1 human ES cell line on synthetic hydrogels under defined conditions. Components include di
A reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before.
Spheroids were assayed in 24-well plate.
A reproducible iPSC-derived human 3D brain microphysiological system, comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before.
MPS with microcirculation mimicking the in vivo transport, which includes continuous exchange of nutrients, constant exposure of the tissue to fresh drug compounds, and removal of metabolic waste products.
The CN Bio LiverChip is a 3D liver tissue model which is continuously perfused for long-term culture. A scaffold allows formation of 3D tissue structures from primary human hepatocytes, through which culture medium is circulated repeatedly via an onboard pneumatic microfluidic pumping system.
The CN Bio LiverChip is a 3D liver tissue model which is continuously perfused for long-term culture. A scaffold allows formation of 3D tissue structures from primary human hepatocytes, through which culture medium is circulated repeatedly via an onboard pneumatic microfluidic pumping system.
A catch-all model to describe treatments applied to an entire cell culture flask.
The cell line cultured in this model will change on a study-by-study basis, as this a generic model used to facilitate data entry of toxicity data.
MatTek’s EpiAirway System consists of normal, human-derived tracheal/bronchial epithelial cells (TBE) which have been cultured to form a multilayered, highly differentiated model which closely resembles the epithelial tissue of the respiratory tract. Histological cross-sections of both the in-vitro tissue and a normal human bronchiole reveal a pseudostratified epithelial structure.
Transmission electron microscopy shows numerous microvilli and cilia on the apical surface of the cultures and confirm the presence of tight junctions. Transepithelial electrical resistance of the tissue is similar to in vivo tissue. Mucins are secreted at the apical surface.
The EpiAirway cultures are grown on cell culture inserts at the air-liquid interface, allowing for gas phase exposure of volatile materials for airway inflammation and irritant studies. This convenient format also allows the facile measurement of transepithelial permeability for inhaled drug delivery studies. The tissues can also be used to investigate mechanisms of bacterial infection of the respiratory tract. These and other studies involving asthma, cytokine responses, or various airway disorders can be performed using the EpiAirway tissue.
EpiDerm (MatTek Corporation, MA, USA) is a reconstructed model of human tissue composed of neonatal foreskin-derived keratinocytes. The keratinocytes are cultured on specially prepared permeable cell culture inserts (Millicell CM, Millipore Corp., Bedford, MA, USA) and achieve advanced levels of differentiation characteristic of the mature epidermis. They are organized in basal, spinous, granular and cornified layers with a high concentration of keratohyalin granules and desmosomes.
EpiDerm Full Thickness (MatTek Corporation, MA, USA; EpiDermFT) is a reconstructed model of human tissue composed of normal, human epidermal keratinocytes (NHEK) and normal, human dermal fibroblasts (NHFB) cultured to form a multilayered model of the human dermis and epidermis. The keratinocytes are cultured on specially prepared permeable cell culture inserts (Millicell CM, Millipore Corp., Bedford, MA, USA) and achieve advanced levels of differentiation characteristic of the mature epidermis. EpiDermFT consists of organized keratin 5 expressing basal cells, involucrin and keratin 10 expressing spinous and granular layers, and cornified epidermal layers analogous to those found in vivo. The dermal compartment is composed of a collagen matrix containing viable normal human dermal fibroblasts (NHDF). The epidermal and dermal layers are mitotically and metabolically active and exhibit in vivo-like morphological and growth characteristics which are uniform and highly reproducible. A well-developed basement membrane is present at the dermal/epidermal junction.
FMi-OOC is a microfluidic organ-on-chip (OOC) device containing primary or immortalized cells (decidua, chorion, and amnion [mesenchyme and epithelium]) from the fete-maternal interface (FMi) and collagen matrix harvested from primary tissue. The FMi-OOC is composed of four concentric circular cell/collagen chambers designed to mimic the thickness and cell density of the FMi in vivo. Each layer is connected by arrays of microchannels filled with type IV collagen to recreate the basement membrane of the amniochorion.
This model is based on the Mimetas 3-lane organoplate. It contains podocytes and glomerular endothelial cells layered in the "glomerular" channel, as well as an empty, "vascular" channel on the other side of the gel. The gel lane is filled with collagen ECM.
The Mattek Epi-Intestinal model is based on human small intestinal cells (ileum) differentiated after being seeded onto permeable supports under air-liquid interface culture conditions. This commercial model has been well characterized and is available in different formats, including a “full thickness” model in which gut cells are grown on top of a smooth muscle epithelial layer, or a “partial thickness” model with just the gut epithelial cells.
Enteroid cysts (three-dimensional [3D] structures) are seeded onto permeable membrane supports and grown to confluency to generate monolayers (two-dimensional [2D] structures). The resulting monolayers allow for controllable access to both apical and basolateral surfaces of the intestinal epithelial cells.The monolayer format, as opposed to the classical Matrigel-embedded 3D cyst culture, allows manipulations both at the apical and basolateral cell surfaces in a compartmentalized manner, thereby broadening options for treatment and collection of specimens (i.e. cells and culture media). It also improves reproducibility in evaluation of outcomes by reducing the variation in cell number and the restricted lumen volume inherent in the cysts cultures.
A simple model where primary J2 intestinal cells are seeding into a 96-well plate format.
This model is used for comparison with JHU's transwell enteroid model
A "2D," 96-well plate based model of Himmelfarb et al.'s Kidney Proximal Tubule organ model. This model simply seeds the RPTEC directly into the wells of the plate. There is no flow.
The kidney tubule MPS exhibits long-term viability, retains polarized expression and function of proteins essential for reabsorptive and secretory transport, responds to physiological stimuli, and performs critical biochemical synthetic activities.
PTECs grown in the MPS polarize with proteins selectively localized to the basolateral and apical aspects of the tubular epithelium and exhibit expected morphological and functional phenotypes of proximal tubule epithelium in vivo out to 28+ days.
MIT's modification of the Kidney Proximal Tubule model to work in the triple channel Nortis Har-v1 (TSC-xxx) device instead of the original Nortis Par (SCC-xxx) device.
An initial version of the KPT MPS model, based on Nortis' prototype Par-V1 device. This model was used in the Weber et al. 2016
The kidney tubule MPS exhibits long-term viability, retains polarized expression and function of proteins essential for reabsorptive and secretory transport, responds to physiological stimuli, and performs critical biochemical synthetic activities.
PTECs grown in the MPS polarize with proteins selectively localized to the basolateral and apical aspects of the tubular epithelium and exhibit expected morphological and functional phenotypes of proximal tubule epithelium in vivo out to 28+ days.
A passive pumping microfluidic chip with up to sixty parallel channels molded with PDMS placed on a plastic/ glass substrate for capture and analysis of immune cells from blood using bright field/ fluorescent microscope imaging.
The Liver Acinus MicroPhysiology System (LAMPS) is a new generation of the liver model developed from SQL-SAL 1.5. The model includes 4-Liver cell types model and the Space of Disse
This model is the Nortis Device equivalent to the 96 MCF7 Metastatic Breast Cancer Co-Culture Model. It contains the 4-cell types from the LAMPS model with the addition of the varioius MCF7 mutant cells
In order to verify growth patterns in the LAMPS microfludic model with the addition of MCF7 breast cancer mutant cells a static plate co-culture model consisting of the 4 cell types of the liver and the addition of the MCF7 mutant cells was created.
Remarkable advances in three-dimensional (3D) cell cultures and organ-on-a-chip technologies have opened the door to recapitulate complex aspects of human physiology, pathology, and drug responses in vitro. The challenges regarding oxygen delivery, throughput, assay multiplexing, and experimental complexity are addressed to ensure that perfused 3D cell culture organ-on-a-chip models become a routine research tool adopted by academic and industrial stakeholders. To move the field forward, we present a throughput-scalable organ-on-a-chip insert system that requires a single tube to operate 48 statistically independent 3D cell culture organ models. Then, we introduce in-well perfusion to circumvent the loss of cell signaling and drug metabolites in otherwise one-way flow of perfusate. Further, to augment the relevancy of 3D cell culture models in vitro, we tackle the problem of oxygen transport by blood using, for the first time, a breathable hemoglobin analog to improve delivery of respiratory gases to cells, because in vivo approximately 98% of oxygen delivery to cells takes place via reversible binding to hemoglobin. Next, we show that improved oxygenation shifts cellular metabolic pathways toward oxidative phosphorylation that contributes to the maintenance of differentiated liver phenotypes in vitro. Lastly, we demonstrate that the activity of cytochrome P450 family of drug metabolizing enzymes is increased and prolonged in primary human hepatocytes cultured in 3D compared to two-dimensional (2D) cell culture gold standard with important ramifications for drug metabolism, drug-drug interactions and pharmacokinetic studies in vitro.
LumeNEXT uses two components - a microfluidic chamber and a removable PDMS rod. The microfluidic chamber is full with collagen that contain different mixes of cells. After collagen polimarization, the rod is removed and endothelial cells can be seed there. This device has a modification in one of the lateral ports. This port is bigger and enable the seed of spheroids. In the large port LumeNEXT device, the stroma and the tumor can be split and analyze independently but maintain the crosstalk.
iPSC-derived cardiomyocytes plated on a micropatterned 96-half-well plate. Micropatterning aligns cells similarly to structure of heart muscle in-vivo.
iCell hepatocytes are seeded into collagen I gel (4mg/mL) at a density of 15,000 cells/uL in the gel channel. HMEC-1 (20,000 cells/uL) and THP-1 (3,000 cells/uL) are pooled and seeded into the fluidic channel. The device is placed on a rocker for gravity-driven perfusion and media is exchanged every 24h.
The OrganoPlate® 2-lane is an advanced microfluidic tissue culture device that contains 96 independent microfluidic chips. Each chip supports an ExtraCellular Matrix (ECM) channel and a perfused medium lane, with no physical barrier in between. A single chip is connected to four wells of the OrganoPlate®: a gel inlet, two medium reservoirs and an observation window. Any number of chips can be used in an experiment. For central nerve system culture without astrocytes no induced perfusion in the system is required.
In cardiomyocytes, pacemaking arises from an interplay between hyperpolarizing and dominating depolarizing currents during the phase 4 depolarization period (the period between repolarization and the rising phase of the subsequent action potential). In the sinoatrial node, the hyperpolarization-induced inward current (HCN isoforms) of cardiac pacemaker cells plays a major role in pacemaking (47). However, in the case of our G-node where stem cell-derived CMs and NRVMs supposedly lack the expression of HCNs, the pacemaking potentials are a result of inward currents produced by Ca2+ cycling (driven by rhythmic releases of intracellular Ca2+ from the sarco/endoplasmic reticulum).
The remaining question was how a region of cells initiate coordinated pacemaking and how this relates to electrical cell-to-cell coupling. The geometrical node design plays a crucial role here because the current being exchanged between individual cells of different membrane potentials is locally accumulated in the membrane capacitance at the edges and is reflected at the tissue edges. The reflection of intracellular currents at the tissue edges synchronizes the spontaneous activity in the structurally isolated small tissues like a G-node and increases their firing rate. The mechanism of reflection at the corners of cultures behave similarly (since downstream impedance is reduced), in particular the anterior corners with acute angles albeit less than in the G-node, and as a result, firing is enhanced in the whole anterior side.
The 3D structures of the bioreactor were modeled using Magics 14 (Materialise, Belgium). The chamber and insert were fabricated using a stereolithography apparatus (EnvisionTec, Germany) employing e-shell 300 as the resin. A multichamber bioreactor was fabricated and fitted into a microfluidic base. An individual bioreactor is composed of a removable insert within a chamber of a well on a 24-well microfluidic plate. The chamber is fixed in place with two O-rings. The osteochondral construct within the insert creates the separation between the upper (chondrogenic) and lower (osteogenic) medium conduits. Opposing gradients of chondrogenic and osteogenic factors and stimulants aid in forming an interface. A single bioreactor is formed by the inset and lid in a well of the plate. Chondrogenic medium (CM) are supplied through upper conduit and osteogenic medium (OM) through the lower conduit.
This model uses the CNBio T12 plate with Transwells (0.4uM). Proximal tubule cells are seeded either in monoculture, or co-culture with endothelial cells, and exposed to flow within the system.
This chip is based on the Mimetas 3-Lane Organoplate. RPTECs are seeded in the "tubular" channel, and HUVECs in the "vascular" channel. 4mg/mL Collagen I gel separates the 2 fluidic layers.
This model represents the RED assay conditions and protocol used when testing serum compound binding for the media used in Columbia's Bone/Bone-Tumor model.Serum protein concentration is at ??%.
This model represents the RED assay conditions and protocol used when testing serum compound binding for the media used in JHU's Intestinal Enteroid model.
This model represents the RED assay conditions and protocol used when testing serum compound binding for the media used in the Kidney Proximal Tubule model.Serum protein concentration is low, at 0.5%.
This model represents the RED assay conditions and protocols used when testing media serum compound binding for the media used in UCal Berkeley Liver Sinusoid model, as well as derived models from Texas A&M.
This model represents the RED assay conditions and protocols used when testing media serum compound binding for the media used in University of Pittsburgh SQL-SAL 1.5 model, as well as derived models from Texas A&M.
Biomimetic model for bone microenvironment. We created a model where different bone marrow cells populations (Bone Marrow Mesenchymal Stem cells, adipocytes, osteoblast, fibroblast, osteoclast, and macrophages) are embedded within a 3D collagen matrix. A lumen structure lined with iPSCs endothelial cells represent blood vessels. The cell media is perfused through the lumen channel.
An MPS device for a contractile force model.
Donor cells are split and expanded to prepare enough stock cultures to prepare myobundles. After 4-5 days of growth and expansion, the human skeletal muscle cells are mixed with non-growth factor reduced Matrigel™, thrombin, fibronectin and human growth media. The cell-containing mixture is placed into a mold that helps form the initial muscle bundles. The myobundles are kept in culture for 5-7 days and then the media is changed to differentiation media. The myobundles are kept in the differentiation medium for another 5-7 days. After differentiation the myobundles are tested for their responsiveness to electrical stimulation by developing a contraction.
The model consists of iPSC-derived keratinocytes form a multilayered epidermis and cornifiedlayer at the surface of the epidermis on a dermis consisting of iPSC-derived fibroblasts, similar to those from human keratinocytes and fibroblasts.
Individual skin constructors are placed into the wells of a 48-well plate.
This is the same base chip as the earlier small airway model, however the top layer has been altered to allow for airflow directly over the cells seeded at ALI (See picture)
Sequentially layered, self-assembly liver (SQL-SAL). This microfluidic model is a single chamber model with 4 liver cells types and three addition/sample locations associated to it (Influent, Chamber, and Effluent).
Modified SQL-SAL 1.0. This 4-liver cell microfluidic model has a single chamber model and has three addition/sample locations associated to it (Influent, Chamber, and Effluent).
TAMU static, 2D version of the SQL-SAL 1.5 Liver MPS model based on standard 96-well plate. The SQL-SQL 1.5 (3D) is a single chamber model and has three addition/sample locations associated to it (Influent, Chamber, and Effluent).
vLAMPS = Vascularized Human Liver Acinus Microphysiological System.
It is a glass based MPS based on Micronit middle layer but kept under static culture on a 60mm petri dish
This is a 3 cell model with human astroglial cells (ATCC CRL-8621) and human brain vascular pericytes (ScienCell #1200) cultured on the basolateral surface of a collagen pre-coated 12mm transwell and human brain microvascular endothelial cells (ScienCell #1000) on the apical surface. Standard Corning® 12mm PET transwells (catalog no. 3462) coated with rat tail collagen I by the user prior to use. Inserts with 3.0µm Pore Polyester (PET) Membrane, 12mm trans wells in 12 well plates.
This is a 3 cell model with human astroglial cells (ATCC CRL-8621) and human brain vascular pericytes (ScienCell #1200) cultured on the basolateral surface of a collagen pre-coated 6.5mm transwell and human brain microvascular endothelial cells (ScienCell #1000) on the apical surface. Corning® BioCoat® Collagen I Inserts with 3.0µm Pore Polyester (PET) Membrane pre-coated with Collagen Type I extracellular matrix, 6.5mm trans wells in 24 well plates.
This is a 3 cell model with human astroglial cells (ATCC CRL-8621) and human brain vascular pericytes (ScienCell #1200) cultured together on the same side of a collagen pre-coated 24mm transwell and human brain microvascular endothelial cells (ScienCell #1000) on the opposite surface of the membrane. Corning® BioCoat® Collagen I Inserts with 3.0µm Pore Polyester (PET) Membrane were purchased pre-coated with Collagen Type I extracellular matrix, 24mm trans wells in 6 well plates.
The molds used in this work were created using acrylic and composed of 5 parts, which form the fabrication mold and prefusion chamber (Fig. S1). The TEBV (2 layers) fabrication mold is formed from parts A, B, and C. Part A housed the mandrels about which TEBVs were made and is used in both the fabrication mold and prefusion chamber. There are four steel hollow mandrels (outer diameter 0.63 mm, inner diameter 0.33 mm) at opposite sides of part A, which are mirror reflections of each other and link the assembled chamber to the perfusion tubing and pump. In the fabrication step, the halves of each mandrel are inserted into the chamber (Part A) and brought into contact with each other (Fig. S1ai and S1bi). Part B forms the top layer of the seeding mold with inlets/outlets and grooves, and part C is the bottom layer of the seeding mold with grooves (Suppl Fig. S1aii and S1bii). The grooves on the top and bottom layers each form four semicircular channels (diameter 2.2 mm, length 24 mm) to be used as molds. Once the high-density collagen containing the hNDFs is added and gelled, parts B and C are removed, and the collagen TEBVs are dehydrated. Then the mandrels are drawn out forming a lumen. Following fixation of the collagen tubing on the mandrels, two new flat covers are added (parts D and E) and the final perfusion chamber is completed. The steel mandrels are used for perfusion, with one side acting as the media inlet, and the other as the outlet.
This “tumor-on-a-chip” platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels.
A monoculture of various cancer cell lines used to provide companion growth data for cancer cell lines that are used in the parent, 3D Vascularized Tumor Model.
Coupling of the vLAMPS (vascularized Human Liver Acinus MPS) and the Pancreatic Islet MPS (PANIS) with a common medium. The connection is carried out through the secretome-rich hepatic chamber of the vLAMPS to the top-chamber of the PANIS. Media is collected from the PANIS bottom chamber and the vascular-chamber of the vLAMPS.
The WAT MPS mimics the physiological environment of adipose tissue with three main elements: a media channel, circular cell chambers, and a microporous membrane in between.
Analogous to the in vivo blood circulation, media travels through the media channel as a vasculature like microcirculation between multiple WAT chambers and constantly transports fresh nutrients and other soluble factors (e.g. drug compounds, cytokines) to and metabolic waste and secreted factors away from the tissue.
The media channel and WAT chambers are connected via small micropores (diameter 3 μm) that act as a perfusion barrier. The perfusion barrier mimics the in vivo endothelial barrier by allowing nutrients, drugs, and other media compounds to diffuse to the tissue while protecting the cells from shear stresses.
The circular geometry of the WAT chambers (diameter 600 μm, height 50 μm) creates a homogeneous supply with nutrients for the entire WAT tissue and enables the direct exchange of soluble factors with the media for each individual cell, which is important as in vivo each adipocyte is attached to at least one capillary.
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