A detailed introduction to recombinant protein drugs (Part Seven)

 

Tablo reader up chevron

A detailed introduction to recombinant protein drugs (Part Seven)

Cell culture and target protein expression

(A) common cells

1. Prokaryotic and eukaryotic cells

(1) Prokaryotic cells

Its main feature is that there are no nuclear nucleus and no nucleoli in the cell, only nucleoli, only pseudonucleus, that is, in a low electron-dense region without clear boundaries, there are naked circular DNA molecules that do not bind to proteins; the main organelles within the structure include 70S-type ribosomes, wireless mitochondria, Golgi apparatus, endoplasmic reticulum, centrosome and other structures; there are cell walls outside the cell membrane, and some flagellar structures, and the cell membrane components are different from eukaryotic cells. Transcription and translation occur simultaneously, and the surrounding plasma membrane contains respiratory enzymes.

E. coli (also known as Escherichia coli) and Bacillus subtilis are generally used generally for recombinant protein expression in Prokaryotic cells. Some people also use lactic acid bacteria (bactobacillus) as host cells to construct recombinant engineered cells to express the protein of interest, but they are rarely used for protein drug preparation process and clinical research project.

(2) Eukaryotic cells

Cells containing eukaryotic nuclei (nucleus surrounded by nuclear membranes) are the basic structure of eukaryotes. In its nucleus, DNA and histones together form a chromosomal structure, and nucleoli can be seen in the nucleus. The cytoplasmic inner membrane system is very developed. There are mainly organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria, lysosome and centrosome, which perform specific biological functions. The cell membrane is a three-layer structure with a bilayer of phospholipids as the backbone, and protein molecules with various biological functions inlaid inside and outside or between them, which together realize the function of material exchange and information inside and outside the cell. Most eukaryotes reproduce sexually through cell mitosis.

The eukaryotic cells used for recombinant protein expression are mainly yeast cells, insect cells, plant cells and mammalian cells. The mammalian cell expression system is the mainstream technology direction for the preparation of modern recombinant protein drugs.

2. Related concepts of mammalian cells

(1) Primary culture cell

Primary cells are cells that have been cultured in vitro immediately after removing cells, tissues or organs from the body. The first 10 generations of cells cultured in vitro are also called primary cells. Directly removed cells can be cultured in vitro after simple separation; tissues or organs must first be mechanically sheared, and then digested and dispersed with trypsin to dissociate the cells in the tissues or organs into individual cells before proceeding to cultivate. The growth rate and difficulty of primary cells from different sources are different, but they cannot be subcultured in vitro for a long time.

Primary cells can be widely used in molecular, cell biology, and basic biomedical research, as well as in the fields of pharmacology and toxicology, precision diagnosis and treatment of biomedical industries.

(2) Secondary cells and cell lines

Secondary cells refer to cells that have been propagated after the primary cell culture has been successfully passaged for the first time, and generally refer to isolated tumor cells or cells that have undergone mutation during cell passage. These cells can be continuously subcultured in vitro, someone also call them immortalized cells.

A cell line is a population of cells that can be stably passaged in vitro, and can also be referred to as a cell line. Cell lines are an important tool for recombinant protein expression and are also widely used in many areas of biomedical research and the biomedical industry.

According to the time of subculture, cell lines can be divided into finite cell lines and infinite cell lines.Finite cell lines can generally be passaged in vitro for 40 to 50 generations, such as diploid cells; infinite cell lines can be passaged and cultured indefinitely in vitro, also known as continuous or infinite cell lines. Most of these cells have undergone heterogeneity. Ploidy is a transformed cell line that may be tumorigenic when allogeneic.

A cell line that is isolated from a certain cell line and differs in character from the original cell line is called a sub line.

(3) Cell strain

In the development of cell biology, the concepts of cell line and cell strain were once mixed. Now (specifically after 2013), most researchers in the industry generally agree with the following views:

The cell line refers generally to general passage cells. From the primary culture or the biologically identified cell line, a single culture with special properties or markers, called a cell line, is obtained by single cell isolation and culture or by a method of proliferation after screening (or called cloning). The special properties or signs of a cell line must be present throughout the culture. When describing a cell line, its special properties or signs must be stated.

Similar to cell lines, cell strains can be divided into finite cell strains and continuous cell strains based on the number of passages. The original cell strain is used to further isolate and cultivate a cell population with different characteristics from the original cell line, which can be called a substrain.

3. Formation of cell lines

Except for blood cells and lymphocytes, most normal mammalian cells are anchor dependent (cells must be attached to a solid or fixed surface to grow, divide, and proliferate), and serum dependent (cells must take in growth factors to grow), contact inhibition (cells in contact with cells, their growth will be inhibited) and morphological dependence (most cells in the body are flat or star-shaped, and have a long fiber network structure) and other properties When cultured in vitro, these conditions can change, causing cells to fail to grow and proliferate normally.

During the in vitro culture of primary cells, under human intervention, the cells crossed the physiological critical point, and phenomena such as chromosome breakage and aneuploidy appeared. They lost the characteristics of normal cells and continued to grow and acquire the ability to proliferate indefinitely. We call this process the transformation of cells, or the formation of cell lines.

By expanding cell characteristics, such as extending the cell cycle, shortening the cell doubling time, reducing the requirements of the cell for culture conditions, and improving the genetic stability of the cell, adding appropriate genetic markers, reducing the secretion of unsafe components, etc., in order to expand the cell culture scale to increase the expression level of engineered cells and achieve an increase in drug yield.

5. Management of cell lines

For primary cells, it is necessary to select a uniform donor, stable conditions such as the location of the material and the type of tissue, and record the relevant information.

For passaged cells, the following information must be recorded in detail:

Tissue source: including the species to which the cell donor belongs (such as human or animal), individual sex, age, organs or tissues (such as tumor tissue and its clinical diagnosis, pathological characteristics, etc.), which have been passaged;

Cell biological properties: general and special biological characteristics of cells, such as general morphology, specific structure, karyotype, growth curve, division index, doubling time, survival rate after freezing and thawing, inoculation rate, etc .;

Culture conditions and methods: the living environment to which the cell line (or cell line) is adapted, such as the culture medium, the type of serum and its amount, the cryopreserved solution, the appropriate pH value, etc .;

Other information: such as non-pollution detection, species detection, immune detection, and cell founder, detector, etc.

Comment Log in or Join Tablo to comment on this chapter...
~

You might like savage's other books...