Researchers Find Key To Growing, Differentiating Human Cells

The finding, announced recently at the American Chemical Society Meeting in San Francisco, may one day aid the production of human organs for transplant.

"We're laying the foundation for artificially growing cells that develop specialized characteristics, just as cells that make up organs naturally do in the body," said Douglas Kniss, professor of obstetrics and gynecology at Ohio State.

A new thermal compression technique allowed the researchers to alter the growth medium inside a fibrous-bed bioreactor and change how cells grow and reproduce. That bioreactor is a tissue-growing device previously developed at Ohio State.

In tests, the researchers were able to coax human placental cells to form clusters and initiate the same kind of chemical changes as stem cells in the body do before they differentiate. Co-investigators on the project include Shang-Tian Yang, professor, and Yan Li, a graduate student, both of chemical engineering; Teng Ma, a postdoctoral researcher in obstetrics and gynecology; and Larry Lasky, associate professor of pathology. Li presented the findings at the American Chemical Society meeting.

Yang designed the bioreactor as a three-dimensional alternative to the flat petri dishes that scientists normally use to culture cells. The device is capable of growing cells for a variety of applications including fermentation, animal cell culture, tissue engineering, and waste water treatment.

Previously, Yang and other chemical engineers at Ohio State grew human tumor cells in the bioreactor to produce large quantities of a protein for cancer research. Kniss and his research group used the bioreactor to grow healthy human placental cells with the eventual goal of testing drugs that a woman can take during pregnancy without harming her fetus.

A forest of microscopic polyester fibers anchor living cells in the bioreactor. This allows cells to grow and reproduce as they do in the body, clinging to the fibers as they would to strands of human proteins. Liquid nutrients course steadily through the bioreactor, mimicking blood flow.

In this latest work, the researchers found that enlarging or shrinking the gaps between the fibers in the bioreactor changes how the cells grow and develop.

To reach that finding, however, the researchers had to invent a way to fix the gaps at a particular size. If they simply compressed the fibrous bed, it would bounce back like a soft sponge. So they tried heating and compressing the polyester at the same time, effectively ironing it into shape.

Li explained that the polyester acts like a solid at room temperature, and a liquid at high temperature. At a specific temperature in between, around 160sF, it exhibits characteristics of both solid and liquid on the molecular level. If they compress the material during this critical time, she said, it retains its shape as well as its special molecular properties.

By precisely controlling pressure, temperature, and compression time, the researchers were able to adjust the size of the gaps.

Kniss likened the fiber structure to a scaffold that supports a piece of sculpture. The thermal compression technique, he said, provides a new method to modify the structure of scaffolds that could one day be used to grow human organs in the laboratory.

"The key to making this technology work is finding a cheap and easy way to mass produce the scaffold," Yang said. "And this is the way to do it."

For the second part of this project, the researchers grew placental cells in two fibrous beds, one with an average gap size of 30 micrometers, and another with an average gap size of 40 micrometers. Placental cells, with an average diameter of about 10 micrometers, provide a good test case because they are similar to the cells that make up the body's organs, Kniss said.

When the fibers were an average of 30 micrometers apart, the cells reproduced very quickly and spread throughout the bioreactor.

But when the fibers were 40 micrometers apart, the cells formed clusters in the open spaces, and initiated chemical functions as they would in the body before differentiating.

Kniss said he could tell the clumps of cells were changing their function when they slowed reproduction and increased their metabolic rate. The cells consumed more glucose and produced more lactic acid. They also began producing estrogen, a key hormone that placental cells normally produce.

Yang explained how the researchers believe gap size affected how the cells grew and behaved. When the gaps between the fibers were small enough for the cells to easily bridge the gap (30 micrometers), they reproduced quickly. That mirrors the initial stage of rapid cell production when organs begin to form in the body. When the spaces between fibers were too large for the cells to cross (40 micrometers), they started to grow on top of each other, as they do in the body before the cells differentiate.

"This thermal compression technique allows us to create gaps of a specific size that will drive cells to reproduce or differentiate, and hopefully both," Kniss said.

"For tissue generation we need fast growth at first, then differentiation later. Coming up with a process where you do both in the laboratory is a challenge," Yang said.

Ma said the reason why clumping enables cells to differentiate is still a biological mystery. He thinks other factors, such as chemical communication between cells, may prompt the cells to differentiate.

Kniss has no immediate plans to use this technology to try to grow entire organs. However, he said that researchers in other labs are currently trying to grow bone, bladder, kidney, liver, and cornea cells using related technologies.

"The underlying principles that we are providing will help those people now, and hopefully in the future will help other people produce whole organs," he said.

For her doctoral thesis, Li is working to grow umbilical chord blood cells. If she is successful, the bioreactor may be used one day to grow cells for bone marrow transplant patients.

This work was funded in part by the National Institutes of Health, and the Interdisciplinary Biomaterials Seed Grants Program and Perinatal Research and Development Fund, both of Ohio State.