Bunsen elaborates: What details do I need to pay attention to in cultivating cells?

Bunsen elaborates: What details do I need to pay attention to in cultivating cells? Cultivating cells is like raising a child, it should be carefully cared for and cared for
with the heart.

Go to see them every day to meet the material needs they need, prevent various small accidents such as lack of water in the incubator, insufficient carbon dioxide, and insufficient temperature, and pay attention to many small details to avoid unnecessary waste of manpower and material resources due to repeated experiments
.

So what small details should be paid attention to in cultivating cells? Let's take a look! 1.
Nutrient source of cell growth The medium of different cells is not the same, for most tumor cells, the nutritional formula is generally: 90% synthetic medium (DMEM, RPMI1640, MEM, etc.
) and 10% fetal bovine serum (FBS); To prevent contamination, 1% dual antibody can be added in due course, and the use of antibiotics should be short-term
.

Q: How to choose a cell culture medium formulation? A: In general, when purchasing cells, there will be a detailed introduction to different cell lines, including the status of growth, the type of culture medium, etc
.

For example, human lung cancer cells A549 can be DMEM medium, and the selection of fetal bovine serum can also be selected as a brand and reliable source of fetal bovine serum, which can provide better nutrients to meet the needs
of cell line growth.

Q: If the cells grow slowly, do I need to increase the serum ratio? A: Yes
.

2.
Environment for cell growth A comfortable and sterile environment is an important foundation for
healthy cell life.

Suitable utensils are needed, different shapes, specifications, and versatile Petri dishes, culture flasks and plates, etc.
, to enter the suitable incubator for culture, such as tumor cells need to grow
in an incubator of 37 °C and 5% CO2.

Q: How to choose a cell culture plate and a Petri dish or flask? A: According to different applications to choose different dishes or volume plates, such as MTS with 96-well plate, cell crawling with 24 wells, flow analysis with 6 empty, etc.
and choose a Petri dish or choose a culture flask, in terms of cell culture state is not much different, mainly the safety factor of the flask is higher, the number will be larger, but the cost is relatively high, so when there is no special requirement, generally use a Petri dish
.

Q: Does the serum have to be inactivated to ensure that the environment is sterile? A: It depends on your application
.

During inactivation, certain components in the serum are destroyed, such as amino acids, vitamins, growth factors, etc
.

Therefore, inactivation of serum will only be considered if your experiment has special requirements for serum
.

If your experiment is sensitive to a component in the serum, it needs to be removed
.

It is common to remove complement proteins from the serum, because complement is involved in cell killing, macrophage and lymphocyte activation, mast cell and platelet histamine release, smooth muscle contraction and other processes, so it is generally necessary to inactivate serum in immunology-related studies and during the culture of muscle cells and stem cells
.

3.
Cell recovery and cryopreservation Cell revival refers to the process
of thawing frozen cells and then re-cultured, and the cells resume growth from the frozen stop growth state.

Q: After cell recovery, why is it difficult to adhere? A: Ignoring the problem of culture media, the main consideration is that the cell cryopreservation is in a poor state or the action is too slow during recovery to cause cell death
.

Remember that the melting rate is important to be fast, and the cryovials can be shaken from time to time to pass through the vulnerable temperature range (-5 to 0 °C)
as soon as possible.

Cell cryopreservation is to place cells at low temperatures (-70 °C ~ 196 °C) to reduce metabolic activity within the cells and limit the preservation of cell viability for long-term storage
.

Q: What are the current formulations used in cell cryocryome? A: At present, cell cryopreservation mostly uses DMSO dimethyl sulfoxide or glycerin as a protective agent, and there are many kinds of formulas of cell cryopreservation solution, such as culture medium: serum: DMSO=7:2:1 or 8:1:1 or 5:4:1, or directly with serum: DMSO = 9:1, generally high concentration of serum helps to maintain cell viability, and the recovery survival rate is above
80% to 90%.

4.
Passage culture of cells Cells continue to proliferate during the culture process, the space of the dish is limited, and the growth of cells too dense will be inhibited, affecting the state of the cell, so we must divide part of the cell into other dishes, so that the cells can grow well
.

Q: Why should I choose logarithmic cells for cell subculture? A: The growth and division of cells generally follows the following patterns: stagnation phase, logarithmic phase (exponential phase), stationary phase (plateau phase) and recession phase
.

To ensure viability, genetic stability, and phenotypic stability, the cells must be kept in the logarithmic phase
.

Generally, cells grow to about 70% to 80% and can be passed on
.

In addition to the joint problems in the above links, there are many small problems in cell culture as follows: Q: How often is the medium changed? A: Normally, the culture medium is reddish
.

If the cells are maintained at pH 6.
5 to 6.
6 and the medium turns yellow, the metabolites in the culture medium have accumulated to a certain amount, the cells will fall off and die, and the fresh culture medium
needs to be replaced.

In general, when the cell growth is strong, it can be changed once every 1 to 2 days, and when the growth is slow, it can also be
changed for 3 to 4 days.

For resuscitation cells, alternate fluid
changes are recommended.

Q: How should the serum be melted? A: Remove the serum from the refrigerator, melt at 2-8 °C, and mix well
by swirling mix from time to time during the melting process.

Dispense well or equilibrate to room temperature before use
.

Q: If the cell morphology is not clear or there is a foreign body, etc.
, what should I do? A: The following operations can be considered: First, discard the old medium, wash with the new medium or PBS 2-3 times, and then start formal digestion and blowing
.

Second, add the blown down cell suspension to a new culture flask
.

Follow up and watch
closely.

Q: Does the flocculent pellet in the serum need to be removed? How to remove? A: There are many reasons that can lead to the formation of flocculent precipitation, one of the common causes is the aggregation of lipoproteins during the melting process
.

This phenomenon generally does not affect product quality
.

Can be centrifuged at 400 g for 5 min, take the supernatant, and then filter
.

Select the appropriate membrane pore size according to your needs, generally 0.
22um PES membrane
.

To avoid membrane blockage, it is recommended to take the supernatant after centrifugation and add it to the medium to filter together instead of directly filtering the serum
.

In short, cell culture is the cornerstone of follow-up experiments, pay attention to various details, strictly adhere to the sterilization process, and protect their own cells, so that they can happily carry out follow-up experiments
.

What are the small details to pay attention to in culturing cells? Bunsen has always regarded quality control as the life of the enterprise, and pursues the continuous improvement
of the competitiveness of the enterprise.

The company has always been adhering to the principle of abiding by discipline and law, strict self-discipline, leniency, and the entrepreneurial spirit of daring to assume as the standard, maintaining and expanding the market with excellent quality and excellent service, and meeting the needs
of customers to a large extent.

Win-win with customers is our development goal
.

Bunsen! Your trusted partner
.

We are willing to cooperate sincerely with you to create a better future
.