Mammary Epithelial Cells and Adipocyte Co-Cultured in a 3-D Matrix: The First Step towards Tissue-Engineered Human Breast Tissue.
Fredrik R.M. Hussa , Gunnar Kratzb
aExperimental plastic surgery department, and bDepartment of reconstructive surgery, Karolinska hospital, Stockholm, Sweden. Cells Tissue Organs, 2001, 169, 361-367
Summary:
Human mammary epithelial cells (HMEC) have been grown and studied in vitro for the past 30 years in order to determine the mechanism involved in breast cancer development. However, the purpose of this study was to determine whether or not tissue engineering could be used to regenerate the human breast. To achieve this goal, HMEC and adipocytes were obtained from a sample biopsy of breast from one individual. These cells were then co-cultured both in a regular cell culture condition as well as in collagen gel to test the ability of the co-culture. The cells have been cultured in a 3-D biological matrix in order to ease the transfer of the regenerated tissue back to patient’s body.
In the culture flask, HMEC grew with characteristic cobblestone shape, which was the typical growth pattern of this type of cells (1a). The pre-adipocytes cells, initially grew as large fibroblast-like cell-shaped, containing no lipid inclusions (1b). With time, these pre-adipocytes obtained their round-shaped characteristic as well as accumulating lipid inclusions (1c). These pre-adipocytes grew radially around the epithelial patches and looked large with diffuse boundaries. Pre-adipocytes grown on the plastic culture eventually differentiated into monolocular adipocytes and ultimately lost their ability to stick to the plastic surface of the culture (1d). Therefore, they floated on the top of the medium and did not stain with oil red Oil in a significant manner.
Under light microscopy, HMEC in 3-D culture were observed to send out protrusions, allowing them to move towards each other and to line up. The lined up rows of the HMEC then interconnected and became thicker to form ductules. A rounded swelling appeared at the end of the ductules that mimicked alveoli. Clusters of cells gathered around ductule structures. Under HE staining, these cells appeared to have nucleal staining but non-staining (empty) cytoplasm. Since this was the characteristic of adipocytes, these clusters of cells were inferred as adipocytes (Figure 2). Most of the orO stained cells appeared to contain small lipid inclusions, and this was an expected characteristic of early-differentiated adipocytes. In this study, it has been shown that HMEC and pre-adipocytes isolated from same individual’s biopsy of breast tissue can be co-cultured in a 3-D matrix. The characteristic growth pattern of ductal structure of HMEC embedded in clusters of adipocytes resembles the in vivo structure of breast tissue.
Significance:
Breast cancer is one of the common types of cancer suffered by both women and men worldwide. It often results in loss of one or both breasts; hence, the reconstruction of human breast is of great importance. This study has shown that human autologous breast tissue, which was regarded as mammary epithelial cells and adipocytes, can be co-cultured in a 3-D matrix in vitro. Consequently, we can use a small sample of an individual’s biopsy of breast tissue and subculture it to create enough cells to generate a biodegradable scaffold that can then be re-implanted into the same patient’s body in order to undergo the reconstruction procedure. Accordingly, in vitro production of the breast tissue can have a wide application in reconstruction plastic surgery. This engineered breast tissue can be integrated with the patient’s own tissue; therefore, this method offers a great advantage over conventional artificial implants such as silicon or saline prosthesis methods.
10 comments:
Why did the researchers choose to co-culture the cells rather than culturing each cell individually?
Did the paper mention anything on effectiveness of growing of HMEC in vivo?
It seems all of these experiments have only been done in vitro. Have there been successful implantations in vivo? Also, the behavior of the pre-adipocyte cells resembles that of adipocytes, but is there a way to confirm that these are indeed adipocytes. After all, it is possible that cells can imitate the behavior of another without actually being the same type of cell.
The paper mentions that "The cells have been cultured in a 3-D biological matrix in order to ease the transfer of the regenerated tissue back to patient’s body". How does it work so? How can that step make the transplantation easier?
"The lined up rows of the HMEC then interconnected and became thicker to form ductules. A rounded swelling appeared at the end of the ductules that mimicked alveoli. Clusters of cells gathered around ductule structures."
This seems ridiculously organized for a simple 3D co-culture. I remember reading about VEG-F making epithelial cells make tube-like structures but this seems to be occurring naturally, in an almost unnatural manner.
And the elephant in the room that everyone has been avoiding:
Are we going to see the replacement of silicone anytime soon?
Ingrid
The whole purpose of this experiment was to determine whether or not the HMEC and Preadipocytes could be co-cultured, since these two cell types are the cells that compose the human breast tissue. They also wanted to look at the growth pattern of these cells in vitro in order to compare that pattern with the characteristics of cells in breast tissue. Thus, by showing that these cells can actually be co-cultured and also by showing that HMEC grew with a characteristic ductule structure within the adipocytes, they proposed that their cell culture resembles the breast tissue in vitro. Also think of what would be the advantage of culturing each cell type individually? Then, maybe these cells could not grow & divide in the presence of the other cell type, and then, the experiment would indicate nothing about the ability to generate the breast tissue in vitro.
No, in this experiment they did not grow HMEC in vivo, only in vitro. They mentioned that previous researchers have cultured HMEC and implanted into nude mice, but only for the purpose of understanding the growth of tumorous cells in vivo, not for the purpose of engineering a breast tissue.
Jesstang
You are right. All these results are for the in vitro experiment and in this paper they have not yet performed the in vivo transfer of the regenerated tissue. The reason is that, they were only able to regenerate a small volume of this breast-like tissue. Since for a larger volume of tissue, they would need more efficient means of nutrient circulation in order to assure the survival of the regenerated cells. They were planing to provide for this circulation by introducing vasculature in in vitro cell culture, or by spares seeding of the matrix. They said that once they implant this regenerated tissue, the matrix will degrade in vivo, and the seeded cells will grow and undergo cell devision leading to formation of neo-vascularization(formation and protrusion of capillary from pre-existing blood vessel) from periphery.
This group used two Cell types, one of which was preadipocytes. To confirm the presence of the Adipocytes, they actually used Oil red O staining. OrO is used to stain the lipids , so it can be used to visualize the adipocytes. Later on based on the acquired morphology of the mature adipocytes, this group identified these cells as the adipocytes containing the OrO stained lipid inclusions ( which was the characteristics of the 'early-differentiated adipocytes')
Oanh
When they grew the two cell types, HMEC and preadipocytes, in the 3D matrix, the HMEC showed a pattern of ductal structures embedded in clusters of adipocytes. This pattern resembled the in vivo structure of breast tissue, and they concluded that this mimicking of the in vivo breast tissue will ease the process of transferring this grown tissue back to patient's body. Although they never actually proceeded with the transferring process in this paper. This experiment as they named it is "The first step towards TE human breast tissue". At the time of this research, 2001, they were still facing the issue of providing enough nutrient to assure the survival of the higher volume of cultured tissue. Thus, their next step after resolving the nutrient issue would be to transfer the engineered tissue.
Yifei
This was the result that they came up with, natural or unnatural I cannot tell without performing their experiment myself. As for silicone, replacing the silicone or saline stuff with autologous tissue would be an optimal end point for this research. When that is going to happen? hopefully some time soon! I'm not too happy about them either. That's why it is important to give our attention to this type of research and to support them.
I definitely like this idea better than the silicon alternative. But like Terry said, vascularization for these organ implants is difficult to do. Over feeding them with hormones to promote angiogenesis is not a viable option either (for example, correlation of VEGF in breast cancer has been strongly suggested in past studies, as Yifei mentioned VEGF). I think you have a good point Azadeh, in saying that we ought to support them because their studies could very well lead to some other discovery which may some day lead to other organ regeneration mechanism (ideally).
Good comment Jessica:)
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