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3D printer stem cells


3D printed tissues and organs without the scaffolding -- ScienceDaily

Engineered tissues and organs have been grown with various degrees of success in labs for many years. Many of them have used a scaffolding approach where cells are seeded onto biodegradable supportive structures that provide the underlying architecture of the organ or tissue desired.

But scaffolds can be problematic -- ultimately, they should degrade and disappear, but timing that decomposition to coincide with the maturation of the organ is tricky, and sometimes degradation byproducts can be toxic. Scaffolds also can interfere with the development of cell-to-cell connections, which are important for the formation of functional tissues.

Now, a research team led by Eben Alsberg, the Richard and Loan Hill Professor of Bioengineering and Orthopaedics at the University of Illinois at Chicago, has developed a process that enables 3D printing of biological tissues without scaffolds using "ink" made up of only stem cells. They report their results in the journal Materials Horizons.

"Our cell only printing platform allows for the 3D printing of cells without a classical scaffold support using a temporary hydrogel bead bath in which printing takes place," Alsberg said.

The micron-scale hydrogel beads allow the nozzle of the 3D printer to move through it and deposit cells with minimal resistance to that nozzle movement or the ejection of the cells. The gel beads support the cells as they are printed and keeps them in place and preserves their shape.

Once the cells are printed into the hydrogel bead matrix, it is exposed to UV light, which cross-links the beads together, in effect freezing them in place. This lets the printed cells connect with each other, mature and grow within a stable structure. The media that bathes the cells flows easily through the cross-linked gel beads and can be changed out as needed to provide fresh nutrients and dispose of waste products made by the cells. The hydrogel beads can be removed through gentle agitation, or controlling their degradation, leaving the intact tissue behind.

"The hydrogel bead bath has unique properties which allow for both printing of the cell-only bioink in complex architectures, and subsequent temporary stabilization of these cell-only structures to allow for cell-cell junctions to form," Alsberg said. "Using chemistry we can then regulate when the beads go away."

The cells Alsberg's team used are stem cells -- those that can differentiate into a wide variety of other cell types. They used the stem cells to 3D print a cartilage ear and a rodent-sized "femur" in the hydrogel bead bath. The cells they printed were able to form stable, cell-cell connections through specialized proteins.

"For the first time, cell-only constructs can be printed in intricate forms that are made up of different cell types without a hydrogel carrier or traditional scaffold that can then be stabilized for a period of a day to weeks. We've demonstrated that cell aggregates can be organized and assembled using this strategy to form larger functional tissues, which may be valuable for tissue engineering or regenerative medicine, drug screening and as models to study developmental biology," Alsberg said.

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3D bioprinting using stem cells

Review

. 2018 Jan;83(1-2):223-231.

doi: 10.1038/pr.2017.252. Epub 2017 Nov 1.

Chin Siang Ong  1 , Pooja Yesantharao  1 , Chen Yu Huang  2 , Gunnar Mattson  1 , Joseph Boktor  1 , Takuma Fukunishi  1 , Huaitao Zhang  1 , Narutoshi Hibino  1

Affiliations

Affiliations

  • 1 Division of Cardiac Surgery, Johns Hopkins Hospital, Baltimore, MD.
  • 2 Division of Cardiology, Johns Hopkins Hospital, Baltimore, MD.
  • PMID: 28985202
  • DOI: 10.1038/pr.2017.252

Review

Chin Siang Ong et al. Pediatr Res. 2018 Jan.

. 2018 Jan;83(1-2):223-231.

doi: 10.1038/pr.2017.252. Epub 2017 Nov 1.

Authors

Chin Siang Ong  1 , Pooja Yesantharao  1 , Chen Yu Huang  2 , Gunnar Mattson  1 , Joseph Boktor  1 , Takuma Fukunishi  1 , Huaitao Zhang  1 , Narutoshi Hibino  1

Affiliations

  • 1 Division of Cardiac Surgery, Johns Hopkins Hospital, Baltimore, MD.
  • 2 Division of Cardiology, Johns Hopkins Hospital, Baltimore, MD.
  • PMID: 28985202
  • DOI: 10.1038/pr.2017.252

Abstract

Recent advances have allowed for three-dimensional (3D) printing technologies to be applied to biocompatible materials, cells and supporting components, creating a field of 3D bioprinting that holds great promise for artificial organ printing and regenerative medicine. At the same time, stem cells, such as human induced pluripotent stem cells, have driven a paradigm shift in tissue regeneration and the modeling of human disease, and represent an unlimited cell source for tissue regeneration and the study of human disease. The ability to reprogram patient-specific cells holds the promise of an enhanced understanding of disease mechanisms and phenotypic variability. 3D bioprinting has been successfully performed using multiple stem cell types of different lineages and potency. The type of 3D bioprinting employed ranged from microextrusion bioprinting, inkjet bioprinting, laser-assisted bioprinting, to newer technologies such as scaffold-free spheroid-based bioprinting. This review discusses the current advances, applications, limitations and future of 3D bioprinting using stem cells, by organ systems.

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Stem cells self-organized and became a material for 3D bioprinting

Scientists have developed a method for printing living tissues on a 3D bioprinter that uses stem cells and one of their most important properties is self-organization. As reported in Nature Materials , stem cells from various tissues placed in favorable conditions self-organized and formed tissues that looked and functioned like full-fledged living tissues.

The formation of tissues in a living organism depends on intercellular contacts and the microenvironment of cells. In the process of development and life activity, cells form around themselves an extracellular matrix - a part of the tissue that serves as a mechanical support and information intermediary for cells. Cells are located in the matrix (respectively, in the tissue) in a spatial relationship characteristic of each organ. To be in the right place at the right time, cells express hundreds of receptors and chemicals that determine how the cell interacts with neighboring cells and the matrix. Thanks to such interactions, cells self-organize - each cell knows where it needs to be in the tissue and what it needs to do.

Until recently, scientists were unable to grow large organelles (more than a centimeter) using 3D bioprinting, either because the cells were too tightly attached to the environment and could not move, or the environment itself did not allow creating the necessary microenvironment. However, Matthias P. Lutolf and colleagues at the Federal Polytechnic School of Lausanne have developed a new 3D bioprinting approach that can solve these problems. The new method, which, among other advantages, allows microscopic work with cell mass and direct observation of the process of printing and growing, uses the self-organization of stem cells as the basis for growing full-fledged organs and tissues. This approach allows you to repeat the natural processes of development of tissues and organs.

To demonstrate the potential and versatility of the new method, the scientists used human small intestine stem cells. The line-printed stem cells were placed on a nutrient medium made of hydrogel with collagen, which is similar in properties to the extracellular matrix. In this environment, cells easily moved and created a fibrous connective tissue structure around themselves, additionally turning the environment into a favorable microenvironment.

A few days later, the cells transformed into a whole and organized epithelial tube 5 to 15 millimeters long, surrounded by a specific matrix, in which scientists found the tissue organization found in the classical organelles of the small intestine. At the same time, scientists note that it was the nutrient medium and the extracellular matrix that had a great influence on the formation of the intestinal tube, which created a favorable microenvironment for cell self-organization. It is noteworthy that intercellular self-organization leveled small printing defects (for example, cell adhesion).

The scientists also managed to grow the epithelium of the mouse small intestine. At first, the stem cells were arranged in a line, but after four or six days, thanks to the self-organization of the cells, a gap appeared in this line, which turned it into a hollow tube. After another one or two days, crypts and villi characteristic of the epithelium of the small intestine were found in the tube, in which scientists found mature differentiated enterocytes, Paneth cells (protective cells that are found only in the small intestine), goblet and enteroendocrine cells. The entire tube responded to external stimuli — Paneth cells released bactericidal granules in response to chemical stimulation, and all cells swelled under the action of forskolin . These reactions show that the new bioprinting technique can produce engineered tissues with physiological responses reminiscent of those in living organisms.

In addition, endothelial cells printed on a mixture with vascular endothelial growth factor (VEGF) formed capillary vessels de novo . Due to favorable conditions (VEGF, loose medium), the formation of capillaries was triggered at the tissue scale, which led to the formation of a vasculature with a continuous lumen.

All of these experiments show that the specific local interactions that govern the self-organization of a small cell block can extend to the tissue level and form different types of tissues: both endothelial and tissues of the internal environment. Using the property of self-organization of stem cells should be an important step towards growing tissues and organs in vitro , because in this case it will be possible to obtain functionally complete organs that can be used for transplantation or drug testing.

Recently, we told that another group of scientists from the same Swiss school has developed a 3D bioprinting method that differs from traditional layer-by-layer 3D printing in that the organ model is created simultaneously over the entire volume in one step, which reduces printing time to several tens of seconds.

Vyacheslav Gomenyuk

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Proposed method for 3D printing of organs from stem cells

Science

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A team of researchers from Scotland have used a new 3D printing technique for the first time to spatially organize human embryonic stem cells (ESCs). So they hope to develop a method for creating organs that is universal for different tissues.

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With the help of 3D printing, it is already possible to produce not only technological parts, but also, for example, elements of prostheses necessary for use in orthopedics or dentistry. So, in early 2012, an 83-year-old woman from the Netherlands, instead of a jaw destroyed by cancer, was implanted with a titanium jaw, printed entirely on a 3D printer. Gazeta.Ru also talked about the American girl Emma, ​​whose exoskeleton elements were printed by using a printer. This summer, an article appeared in which researchers proposed a technique printing of the liver , which allows cells to grow on a framework of sugar tubes.

Scottish scientists have taken another step towards creating full-fledged organs using 3D printing.

They have developed a universal technique for 3D printing tissues using stem cells, which can then be reprogrammed into any desired cells in organs and tissues.

Their work, published in the journal Biofabrication , gives hope that full printing of organs and other biological structures will soon become possible.

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The

3D bioprinter is a biological variation of reprap technology, a device capable of creating organs and tissues by layering cells on top of each other. The first serial bioprinter was created in December 2009 by the American company Organovo and the Australian company Invetech. In contrast to the classical method of growing organs, bioprinting does not require a scaffold on which cells are “settled”, which is an indisputable advantage, since the scaffold can become the initiator of inflammation of the created organ or tissue.

The bioprinter uses two types of "ink" - cells of various types and auxiliary materials (collagen, growth factors, supporting hydrogel), designed to strengthen the created structure until natural bonds form between cells.

Previously, for bioprinting, it was necessary to first grow cultures of cells from which an organ would be created. The cell culture was cut into small balls - spheroids. Spheroids from several separately grown cultures were artificially "planted" side by side. The advantage of the new method is that embryonic stem cells (ESCs) that can develop into any tissue are used for “printing”.

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One of the authors of the study, Dr. Will Wenmiao Shu of Heriot-Watt University, says: “To our knowledge, this is the first time that human embryonic stem cells have been printed. The generation of 3D structures from ESCs will allow us to create more accurate models of human tissues, which is important in the development of drugs in vitro and the study of their toxicity. Since most medicines are targeted at humans, it makes sense to use human tissues.”

In the future, this new bioprinting method can be used to create artificial organs and tissues ready for transplantation in patients with various diseases.

In the study, scientists at Heriot-Watt University, in collaboration with Roslin Cellab, used a flap printing technique adapted for delicate stem cell work. ESCs were loaded into two separate tanks and then applied to the plate according to a pre-prepared pattern. After printing the ESC, a number of tests were carried out in order to understand how effective the new method is. For example, the researchers tested whether ESCs remained alive after being printed and whether they retained the ability to differentiate into different cell types. They also determined the concentration, damage, and other characteristics of the "printed" cells to assess the accuracy of the valve method.

“When using the valve method, stem cell printing is controlled by pneumatic pressure and controlled by the opening and closing of a micro-valve. The number of cells used can be precisely controlled by changing the nozzle diameter, air inlet pressure, or valve opening time,” says Dr. Shu.

“We have found that the valve printing method is soft enough to maintain high stem cell viability and precise enough to produce uniform sized spheroids. And most importantly, the printed ESCs retain the ability to pluripotency - the ability to differentiate into any other cell types,” he said.

There is a lot of attention in the field of regenerative medicine for ESC. They are obtained from embryos in the early stages of development to produce "stem cell lines" capable of constantly growing and differentiating into any type of human cell. Jason King, Business Development Manager at Roslin Cellab, comments: “This scientific development we hope and believe will have important implications for reliable animal-free drug testing and, in the long term, organ creation and on-demand transplantation.” without the need for donation and without the problems associated with immune suppression and possible rejection. ”

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