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3D cell culture

3D cell culture

3D cell culture techniques use engineering to provide 3D environments for cells to grow. Cells can be attached to or embedded in scaffolds engineered from biological or synthetic materials or grown in conditions that promote cells to self-organize into 3D structures.

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Edits on 5 Jul, 2022
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The drive to develop 3D cell culture comes from the understanding that cells grown in culture in 2D dishes do not receive all of the environmental cues received in a living 3D environment. Cells look and behave differently in 2D and 3D systems . Differences in the ways cells behave and communicate with each other in 2D cultures versus animal models is thought to underlie some of the discrepancies in research data between cell cultures and animal models .

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The stem cell niche, the place where stem cells reside within tissues, is a place where stem cells interact with the extracellular matrix (ECM), other cells and growth factors and receive biophysical and biochemical signals that control behaviors such as migration, cell division and differentiation into specialized tissues . Control of stem cell fate through engineered microniches that mimic aspects of the native stem cell niches are important for the advancement of tissue engineering and stem cell therapies .

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The initial approach in 3D tissue engineering was to seed cells onto a prefabricated scaffold. Various scaffolds have been developed from synthetic polymers such as poly-lactic acid, poly-glycolic acid, poly-lactic-co-glycolic acid and poly-caprolactone upon which cells can be seeded . Replicating the microstructural characteristics is a challenge with this approach. Decellularized scaffolds, prepared by removing cells from natural structures like tissues, organs and plants have been used to support the growth of mammalian cells.

...

Hydrogels are porous, soft, polymeric networks which absorb large amounts of water or biological fluids and can simulate living tissue . These can be made with synthetic polymers and natural biomaterials like collagen, fibrin, gelatin, alginate, hyaluronic acid, chitosan, agarose, dextran and Matrigel . Substances like collagen, fibrin and hyaluronic acid are components of the extracellular matrix (ECM), which is the natural scaffold cells are surrounded by in living tissues . Matrigel is the trade name for a solubilized extracellular matrix prepared from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, and it is widely used in 2D and 3D cell culture .

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Hydrogels comprised of synthetic polymers, while lacking endogenous factors that cells encounter in tissues, can be designed with specific physical and chemical properties . For example, since the ECM in living organisms is dynamic and cells respond to ECM stiffness, mechanically dynamic hydrogels have been developed which can stiffen and soften . Synthetic hydrogels allow long term cell culture and are very reproduceable.

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Microgels can be patterned in an array of shapes. They can encapsulate single cells in a 3D microenvironment and have been used to keep single human mesenchymal stem cells alive for up to 4 weeks . The stiffness of the microgels was shown to have an effect on differentiation into bone lineages .

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Microfluidic devices incorporate tiny channels into 3D cell culture systems to perfuse cells with oxygen and nutrients and to remove waste . Microfluidic devices can mimic shear forces that blood vessel endothelial cells would encounter in a living organism . Drugs and biological molecules like growth factors can be supplied through these devices. Seeding fabricated microfluidic networks with human umbilical vein endothelial cells (HUVECs) lead to sprouting and formation of vasculature .

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3D cellular aggregates can form without a scaffold. Hanging-drop technique, invented by Robert Koch in the 1880s, is the culturing of cells in suspended droplets which forces them to aggregate . 3D spheroids can be generated and have been scaled up to 384-hanging-drop arrays. Liquid overlay can generate 3D microtissues on non-adherent surfaces . There are size limitations since nutrients and oxygen must diffuse into the microtissues, and necrotic centers can occur in large spheroids. Micro-molds and patterned microplates control size and consistency . More advanced materials and methods involve spinner flasks, rotating wall vessel bioreactors and microfluidic systems . In addition, physical stimuli like ultrasound traps, electric fields, magnetic forces or affinity between avidin and biotin are used to generate heterospheroids made of more than one cell type .

...

In 1989, mouse mammary cultures grown on a laminin-rich extracellular matrix (ECM), derived from murine tumor, but known as Matrigel today, differentiated into self-organized alveolar structures . The self-organizing capacity of cells on the laminin-rich ECM, Matrigel allows the generation of organoids, 3D organ-like structures . Organoids can be generated from primary tissue, cell lines, adult stem cells (ASCs) and pluripotent stem cells (PSCs). Intestine, colon, liver, pancreatic, lung, stomach, brain, retina, prostate, fallopian tube, mammary gland, taste buds, salivary glands and oesophagus have been developed. They are used in disease modelling, drug testing, biobanking and host-pathogen interaction studies.

...

Hans Clevers’ research group at the Hubrecht Institute, The Netherlands, developed miniguts, intestine organoids with outgrowths areas that mimic the environment of the intestinal crypt, known as a stem cell niche that supports the survival of intestinal stem cells . Clevers developed a personalized drug test for Cystic Fibrosis (CF) using miniguts derived from a patients own cells . CF mutations affect mucous-secreting cells, affecting the lungs and the intestines . There are many different mutations causing CF and certain CF drugs may work on only a subset of patients so growing miniguts with from individual CF patients allows the testing of expensive drugs that insurers may be reluctant to cover . Clevers is part of the group that founded the non-profit Hubrecht Organoid Technology (HUB) to market applications .

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Organoid models for normal and neoplastic mouse and human pancreatic tissues have been established to study tumor progression .

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Lung organoids were developed to model lung development using PSCs. Using growth factors and signaling molecules known to act during embryonic development, most cell types in the respiratory system can be generated from human PSCs .

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Neural differentiation occurs spontaneously in embryonic stem cell (ESC) culture when certain signalling pathways are inhibited . Serum-free floating culture of embryoid body (EB)-like aggregates with quick re-aggregation (SFEBq) is a technique for generating brain organoids in the lab developed by Yoshiki Sasai at the RIKEN Center for Developmental Biology, Japan . Human and mouse ESCs cells are isolated and re-aggregated and then maintained in serum-free growth medium with low levels of growth factors. When transferred onto adhesion plates, they self-organize, forming a lumen and various brain structures.

...

3D printing can be used to arrange living cells and non-cellular material in 3D space to mimic human tissue . Computer aided design (CAD) technologies combined with MRI or CT scans allow bioprinting of complex structures at nanoscale to microscale scale economically and efficiently . When the aim is to transplant bioprinted tissues or organs into a host, suitable bioreactors that mimic the living environment will be needed for incubation while tissues fuse and mature prior to implantation .

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For inkjet bioprinting, bioinks can be pre-polymers with or without cells. Human mesenchymal cells (MSCs) were printed with bioactive glass and hydroxyapatite polymerized in a poly(ethylene) glycol dimethacrylate (PEGDMA) scaffold . Extrusion bioprinting, also known as bioplotting, is suitable for printing viscous materials and is deposited as continuous cylindrical lines rather than as droplets . For printing cells or encapsulated cells, the nozzle needs to be wide enough not to damage them, which can limit resolution.

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Laser-assisted bioprinting (LAB) does not use nozzles and can print a wide range of materials such as cells, cell-encapsulate materials and hydrogels . Lasers irradiate a ribbon which holds cells or biomaterials, causing the liquid biological materials to evaporate and drop onto the receiving substrate, which contains biopolymer or cell culture medium . LAB is able to print high resolution from pico to micro scales but is low throughput and costly . Many cell types including human dermal fibroblasts, breast cancer cells, rat neural cells have been printed with laser-based technology . Fabricated skin for skin grafts using fibroblasts and keratinocytes have been successful in animal studies .

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Natural polymers such as gelatin, collagen, alginate, chitosan, hyaluronic acid and agarose are used in bioinks . Decellularized extracellular matrix derived from tissues can be used as bioink to support cell function . Tissue-specific decellularized extracellular matrix have been used to support adipose, cartilage and heart tissues . Cardiomyocytes encapsulated in alginate and gelatin composite have been used in inkjet printing to construct cardiac tissue . Similarly, a heart valve with porcine aortic valve cells and smooth muscle cells in alginate a composite alginate/gelatin hydrogel showed good cell viability .

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Compared with natural polymers, synthetic polymers have more consistent chemical and mechanical properties . Mechanical properties are better for synthetic polymers, and when conjugated with peptides or adhesive proteins to provide biomimetic cues, cells can interact with the biomaterial in a physiologically relevant manner . Growth factors can provide necessary cues for proper growth of tissues. Calcium phosphate, tri-calcium phosphate, hydroxyapatite, poly-lactic acid (PLA), poly-glycolic acid (PGA), poly(lactic-co-glycolic acid) and poly-caprolactone (PCL) are used in bioprinting. PLA, PGA and PCL are biocompatible, biodegradable, strong and can accelerate bone repair and inhibit inflammation .

Edits on 24 Nov, 2020
Charlie Hilton
Charlie Hilton edited on 24 Nov, 2020
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3D cell culture

3D cell culture techniques use engineering to provide 3D environments for cells to grow. Cells can be attached to or embedded in scaffolds engineered from biological or synthetic materials or grown in conditions that promote cells to self-organize into 3D structures.

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Edits on 3 Dec, 2018
Meredith Hanel
Meredith Hanel edited on 3 Dec, 2018
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Liver organoids were initially generated to study liver development. They were generated using pluripotent stem cell (PSC) derived hepatocytes cultured with mesenchymal stem cells and endothelial cells on a plate coated with Matrigel . These liver organoids contained blood vessels which connected with host mouse when transplanted. Mice with drug-induced liver failure recovered when they received these transplanted liver buds.

Edits on 25 Sep, 2018
Meredith Hanel
Meredith Hanel edited on 25 Sep, 2018
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While mammalian cells have traditionally been grown on two-dimensional tissue culture dishes, 3D cell culture techniques use engineering to provide 3D environments for cells to grow. Cells can be attached to or embedded in scaffolds engineered from biological or synthetic materials or grown in conditions that promote cells to self-organize into 3D structures. 3D cell culture techniques are used to create tissue models for studying disease and testing treatments and also to engineer tissues engineer tissues for transplantation.

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Edits on 14 Sep, 2018
Meredith Hanel
Meredith Hanel edited on 14 Sep, 2018
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The initial approach in 3D tissue engineering was to seed cells onto a prefabricated scaffold. Various scaffolds have been developed from synthetic polymers such as poly-lactic acid, poly-glycolic acid, poly-lactic-co-glycolic acid and poly-caprolactone upon which cells can be seeded . Replicating the microstructural characteristics is a challenge with this approach. Decellularized scaffolds, prepared by removing cells from natural structures like tissues, organs and plants have been used to support the growth of mammalian cells.

Edits on 8 Sep, 2018
Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 8 Sep, 2018
Edits made to:
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Compared with natural polymers, synthetic polymers have more consistent chemical and mechanical properties . Mechanical properties are better for synthetic polymers, and when conjugated with peptides or adhesive proteins to provide biomimetic cues, cells can interact with the biomaterial in a physiologically relevant manner . Growth factors can provide necessary cues for proper growth of tissues. Calcium phosphateCalcium phosphate, tri-calcium phosphate, hydroxyapatite, poly-lactic acid (PLA), poly-glycolic acid (PGA), poly(lactic-co-glycolic acid) and poly-caprolactone (PCL) are used in bioprinting. PLA, PGA and PCL are biocompatible, biodegradable, strong and can accelerate bone repair and inhibit inflammation .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 8 Sep, 2018
Edits made to:
Article (+9/-9 characters)
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3D cellular aggregates can form without a scaffold. Hanging-drop technique, invented by Robert Koch in the 1880s, is the culturing of cells in suspended droplets which forces them to aggregate . 3D spheroids can be generated and have been scaled up to 384-hanging-drop arrays. Liquid overlay can generate 3D microtissues on non-adherent surfaces . There are size limitations since nutrients and oxygen must diffuse into the microtissues, and necrotic centers can occur in large spheroids. Micro-molds and patterned microplates control size and consistency . More advanced materials and methods involve spinner flasks, rotating wall vessel bioreactors and microfluidic systems . In addition, physical stimuli like ultrasound traps, electric fields, magnetic forces or affinity between avidin and biotin are used to generate heterospheroids made of more than one cell typecell type .

Edits on 7 Sep, 2018
Dawson Sewell
Dawson Sewell edited on 7 Sep, 2018
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The drive to develop 3D cell culture comes from the understanding that cells grown in culture in 2D dishes do not receive all of the environmental cues received in a living 3D environment. Cells look and behave differently in 2D and 3D systems . Differences in the ways cells behave and communicate with each other in 2D cultures versus livinganimal organismsmodels is thought to underlie some of the discrepancies in research data obtained throughbetween cell cell culturecultures versusand animal models .

...

Hans Clevers’ research group at the Hubrecht Institute, The Netherlands, developed miniguts, intestine organoids with outgrowths areas that mimic the environment of the intestinal crypt, known as a stem cell niche that supports the survival of intestinal stem cells . Clevers developed a personalized drug test for Cystic Fibrosis (CF) using miniguts derived from a patients own cells . CF mutations affect mucous-secreting cells, affecting the lungs and the intestines . There are many different mutations causing CF and certain CF drugs may work on only a subset of patients so growing miniguts with from individual CF patients allows the testing of expensive drugs that insurers may be reluctant to cover . Clevers is part of the group that founded the non-profit Hubrecht Organoid Technology (HUB) to market applications .

...

3D printing can be used to arrange living cells and non-cellular material in 3D space to mimic human tissue . Computer aided design (CAD) technologies combined with MRI or CT scans allow bioprinting of complex structures at nanoscale to microscale scale economically and efficiently . When the aim is to transplant bioprinted tissues or organs into a host, suitable bioreactors that mimic the living environment will be needed for incubation while tissues fuse and mature prior to implantation .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 7 Sep, 2018
Edits made to:
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Hans Clevers’ research group at the Hubrecht Institute, The NetherlandsNetherlands, developed miniguts, intestine organoids with outgrowths areas that mimic the environment of the intestinal crypt, known as a stem cell niche that supports the survival of intestinal stem cells . Clevers developed a personalized drug test for Cystic Fibrosis (CF) using miniguts derived from a patients own cells . CF mutations affect mucous-secreting cells, affecting the lungs and the intestines . There are many different mutations causing CF and certain CF drugs may work on only a subset of patients so growing miniguts with from individual CF patients allows the testing of expensive drugs that insurers may be reluctant to cover . Clevers is part of the group that founded the non-profit Hubrecht Organoid Technology (HUB) to market applications .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
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Hydrogels are porous, soft, polymeric networks which absorb large amounts of water or biological fluids and can simulate living tissue . These can be made with synthetic polymers and natural biomaterials like collagen, fibrin, gelatin, alginate, hyaluronic acidhyaluronic acid, chitosan, agarose, dextran and Matrigel . Substances like collagen, fibrin and hyaluronic acid are components of the extracellular matrix (ECM), which is the natural scaffold cells are surrounded by in living tissues . Matrigel is the trade name for a solubilized extracellular matrix prepared from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, and it is widely used in 2D and 3D cell culture .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+5/-5 characters)
Article

Neural differentiation occurs spontaneously in embryonic stem cell (ESC) culture when certain signalling pathways are inhibited . Serum-free floating culture of embryoid body (EB)-like aggregates with quick re-aggregation (SFEBq) is a technique for generating brain organoids in the lab developed by Yoshiki Sasai at the RIKEN Center for Developmental Biology, JapanJapan . Human and mouse ESCs cells are isolated and re-aggregated and then maintained in serum-free growth medium with low levels of growth factors. When transferred onto adhesion plates, they self-organize, forming a lumen and various brain structures.

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+13/-13 characters)
Article

The stem cell niche, the place where stem cells reside within tissues, is a place where stem cells interact with the extracellular matrix (ECM), other cells and growth factors and receive biophysical and biochemical signals that control behaviors such as migration, cell divisioncell division and differentiation into specialized tissues . Control of stem cell fate through engineered microniches that mimic aspects of the native stem cell niches are important for the advancement of tissue engineering and stem cell therapies .

Edits on 6 Sep, 2018
Carla Faraguna
Carla Faraguna approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
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Hans CleversHans Clevers’ research group at the Hubrecht Institute, The Netherlands, developed miniguts, intestine organoids with outgrowths areas that mimic the environment of the intestinal crypt, known as a stem cell niche that supports the survival of intestinal stem cells . Clevers developed a personalized drug test for Cystic Fibrosis (CF) using miniguts derived from a patients own cells . CF mutations affect mucous-secreting cells, affecting the lungs and the intestines . There are many different mutations causing CF and certain CF drugs may work on only a subset of patients so growing miniguts with from individual CF patients allows the testing of expensive drugs that insurers may be reluctant to cover . Clevers is part of the group that founded the non-profit Hubrecht Organoid Technology (HUB) to market applications .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+11/-11 characters)
Article

3D cellular aggregates can form without a scaffold. Hanging-drop technique, invented by Robert KochRobert Koch in the 1880s, is the culturing of cells in suspended droplets which forces them to aggregate . 3D spheroids can be generated and have been scaled up to 384-hanging-drop arrays. Liquid overlay can generate 3D microtissues on non-adherent surfaces . There are size limitations since nutrients and oxygen must diffuse into the microtissues, and necrotic centers can occur in large spheroids. Micro-molds and patterned microplates control size and consistency . More advanced materials and methods involve spinner flasks, rotating wall vessel bioreactors and microfluidic systems . In addition, physical stimuli like ultrasound traps, electric fields, magnetic forces or affinity between avidin and biotin are used to generate heterospheroids made of more than one cell type .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+13/-13 characters)
Article

Neural differentiation occurs spontaneously in embryonic stem cell (ESC) culture when certain signalling pathways are inhibited . Serum-free floating culture of embryoid body (EB)-like aggregates with quick re-aggregation (SFEBq) is a technique for generating brain organoids in the lab developed by Yoshiki SasaiYoshiki Sasai at the RIKEN Center for Developmental Biology, Japan . Human and mouse ESCs cells are isolated and re-aggregated and then maintained in serum-free growth medium with low levels of growth factors. When transferred onto adhesion plates, they self-organize, forming a lumen and various brain structures.

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+15/-15 characters)
Article

Hans Clevers’ research group at the Hubrecht Institute, The Netherlands, developed miniguts, intestine organoids with outgrowths areas that mimic the environment of the intestinal crypt, known as a stem cell niche that supports the survival of intestinal stem cells . Clevers developed a personalized drug test for Cystic FibrosisCystic Fibrosis (CF) using miniguts derived from a patients own cells . CF mutations affect mucous-secreting cells, affecting the lungs and the intestines . There are many different mutations causing CF and certain CF drugs may work on only a subset of patients so growing miniguts with from individual CF patients allows the testing of expensive drugs that insurers may be reluctant to cover . Clevers is part of the group that founded the non-profit Hubrecht Organoid Technology (HUB) to market applications .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+9/-9 characters)
Article

In 1989, mouse mammary cultures grown on a laminin-rich extracellular matrix (ECM), derived from murine tumor, but known as Matrigel today, differentiated into self-organized alveolar structures . The self-organizing capacity of cells on the laminin-rich ECM, Matrigel allows the generation of organoidsorganoids, 3D organ-like structures . Organoids can be generated from primary tissue, cell lines, adult stem cells (ASCs) and pluripotent stem cells (PSCs). Intestine, colon, liver, pancreatic, lung, stomach, brain, retina, prostate, fallopian tube, mammary gland, taste buds, salivary glands and oesophagus have been developed. They are used in disease modelling, drug testing, biobanking and host-pathogen interaction studies.

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+11/-11 characters)
Article

3D printing3D printing can be used to arrange living cells and non-cellular material in 3D space to mimic human tissue . Computer aided design (CAD) technologies combined with MRI or CT scans allow bioprinting of complex structures at nanoscale to microscale scale economically and efficiently . When the aim is to transplant bioprinted tissues or organs into a host, suitable bioreactors that mimic the living environment will be needed for incubation while tissues fuse and mature prior to implantation .

Jude Gomila
Jude Gomila approved a suggestion from Golden's AI on 6 Sep, 2018
Edits made to:
Article (+4/-4 characters)
Article

In 1989, mouse mammary cultures grown on a laminin-rich extracellular matrix (ECM), derived from murine tumor, but known as Matrigel today, differentiated into self-organized alveolar structures . The self-organizing capacity of cells on the laminin-rich ECM, Matrigel allows the generation of organoids, 3D organ-like structures . Organoids can be generated from primary tissue, cell lines, adult stem cells (ASCs) and pluripotent stem cells (PSCs). Intestine, colon, liver, pancreatic, lunglung, stomach, brain, retina, prostate, fallopian tube, mammary gland, taste buds, salivary glands and oesophagus have been developed. They are used in disease modelling, drug testing, biobanking and host-pathogen interaction studies.

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