Extraction of Primary Cells from Tissue

introduction

Primary culture is a type of cell culture system where cells are directly obtained from tissue. Primary culture begins with a biopsy of tissue or an organ, typically through the excision of a piece measuring 1 to 3 centimeters. In tissue organization, cells have basal membrane connections and cell-cell junctions that must first be disrupted.

To perform this process, various cell isolation methods are used, such as enzymatic or chemical digestion methods involving different proteolytic enzymes like trypsin or collagenase, as well as mechanical separation methods like tissue dissection with surgical blades. After thoroughly washing the tissues and removing the connections, the resulting suspension is purified through centrifugation and transferred to culture vessels. Subsequently, many cells gradually detach from the tissue and adhere independently to the bottom of the flask.

Once healthy cells with appropriate morphology are extracted from the tissues, the primary culture process is completed. However, in addition to the specific cells initially sought in primary cultures, various cell types, especially fibroblasts, are also observed to transfer to the culture flasks. To eliminate contaminating cells, various selective culture media can be used, and the stemness of the extracted cells can be confirmed by examining positive and negative surface markers.

Extraction of Stem Cells

Stem cells are located in various parts of the body. In adults, these cells are found in bone marrow, adipose tissue, brain, dental roots, blood, liver, heart, skeletal muscles, etc. The best technique is one that allows for the maximum extraction of stem cells with minimal damage.

One of the most important criteria in selecting the extraction method and target tissue is the intended use of the stem cells. For example, in direct transplantation of stem cells from a living donor to another individual, it is necessary to use bone marrow stem cells. Although this method causes significant pain to the donor, after performing HLA typing confirmation tests, no method other than bone marrow extraction remains. However, in research models, discarded human adipose tissue produced during liposuction cosmetic procedures can be utilized.

The stem cells widely extracted and cultured by researchers include embryonic stem cells (ESCs), which are pluripotent, induced pluripotent stem cells (iPSCs), and adult stem cells, which are multipotent. Naturally, there are many other diverse and extensive types of stem cells in the human body that are of high research value. However, these three types of cells are among the most commonly used and are simpler to extract.

Types of Stem Cells and Their Origins

Embryonic stem cells were among the earliest cells to be extracted and cultured in the late 20th century. In Iran, individuals such as Dr. Ahmadreza Bahrami and Dr. Maryam Moghaddam Matin were among the first to extract and culture these cells in the early years of the 21st century.

Although embryonic stem cells have a high capability for differentiation into various cell types, their extraction and culture are challenging and raise significant ethical issues. It is important to note that to extract embryonic stem cells, a blastocyst-stage embryo must be obtained. The resulting cell colony must then be transferred to a supportive layer of cells known as MEF (Mouse Embryonic Fibroblasts) for culture. Additionally, the culture medium used for embryonic stem cells can be quite costly, as various growth factors are required for their growth.

Moreover, confirmation tests for embryonic stem cells can be very rigorous and expensive. While the high differentiation potential of these cells can be seen as an advantage, this same high potential increases the risk of teratoma formation and malignancy upon transplantation.

The ethical concerns surrounding the extraction of embryonic stem cells led a Japanese scientist in 2006 to produce these cells from differentiated cells. One of the methods for producing ESCs involves transferring the nucleus of a differentiated cell into an enucleated egg cell and implanting it into a surrogate uterus. This means that to create a patient-specific ESC, complete nucleated cells must be used, and the 2n nucleus of that cell is implanted into an egg.

The haploid egg, which has been artificially converted into a diploid zygote, is then implanted in the surrogate uterus and subsequently extracted and cultured at the morula stage.

Through the efforts of a Japanese scientist named Shinya Yamanaka, genes that function as master genes were identified, and by introducing these genes, induced pluripotent stem cells (iPSCs) can be produced that function similarly to embryonic stem cells. In this method of extraction and production, a differentiated fibroblast cell is obtained from an individual and cultured.

Then, using various methods such as transduction, transfection, and epigenetic modification, the expression of a set of genes including Klf4, Sox2, Oct3/4, and c-Myc is increased, leading to dedifferentiation and conversion of the functional cell into an induced pluripotent stem cell (iPSC).

Today, one of the most widely used types of stem cells around the world is adult stem cells. When referring to adult stem cells, it often pertains to mesenchymal stem cells. These cells are distributed throughout the body and have various niches, with some niches exhibiting a more prominent presence.

For example, in an adult individual, these cells are most abundant in the bone marrow, adipose tissue, and dental roots. They also have a higher presence in tissues related to the embryo, such as the umbilical cord. Therefore, in extracting these cells, it is essential to consider the appropriate tissue for maximum extraction while minimizing harm to the donor.

Although the process of extracting bone marrow tissue is very painful and causes significant harm to the individual, due to the loose intercellular connections, their extraction from this tissue is relatively easier. After several washes and using substances that create a gravitational gradient (such as ficoll), mesenchymal stem cells can be purified.

In other tissues such as adipose tissue or umbilical cord, where mesenchymal stem cells are present, the intercellular connections are stronger, and these connections must be broken to extract the cells. The extracellular matrix in these tissues is primarily composed of collagen type I. Therefore, to break the integrins attached to collagen, trypsin can be used, and collagenase type 1 can be used to break down collagen fibers.

After extraction, these tissues must be thoroughly washed to ensure complete removal of blood. They are then treated with enzymes that break down intercellular connections to release the cells completely from the tissue. After enzyme treatment, to neutralize its effects, a culture medium containing FBS is added, and the mixture is centrifuged. In adipose tissue, a deposit known as Stromal Vascular Fraction (SVF) is formed, which can be cultured to purify mesenchymal stem cells.