As we all know, the types of cells cultured in the laboratory can be roughly divided into two types: adherent cells and suspension cells, and only a few types of cells exist in both states.
Suspension cells refer to a type of cells that can grow in a suspended state in a medium without relying on supports, such as lymphocytes and most blood system-derived cells. (such as mouse leukemia cells WEHI-3, human leukemia cells K-562, HL-60). In the industry, more and more adherent cells have been domesticated and cultured in full suspension. At present, SP2/0, NS0, CHO, BHK and HEK293 cells are mainly used in the industry, and they are mainly used for recombinant protein production; the passage cells commonly used in veterinary biological products mainly include BHK21 cells cultured in full suspension, MDCK cells; and Vero cells, PK cells, ST cells, Marc145 cells, etc. with the help of microcarrier suspension culture, each cell has different characteristics.
Suspension cells
Suspension cells "thp-1" 200x
In general, suspension cells are easier to handle than adherent cells because no trypsinization is required for passage of suspension cells. But this does not mean that suspension cells are easier to grow than adherent cells. For novices, it is more challenging. There are always various problems: for example, why do dead cells and cell differentiation always appear; it is agreed that the passage is easy, but the result will be differentiation; problems appear again after cryopreservation and recovery!
Next, let's discuss some key points about culturing suspension cells~
Point 1: Enough patience, it is very necessary
Many suspension cells have relatively poor viability when they are just recovered from cryopreservation. At this time, we need to be patient enough to wait for the cells to complete their self-repair and enter the logarithmic growth phase. For example, THP-1 (human monocytic leukemia) often needs 7-10 days of recovery period from thawing to normal proliferation; Jurkat, Clone E6-1 (human T lymphocytic leukemia cells) also need 5-7 days after recovery. Return to the state before freezing. Novices may give up training during this period, so please be more patient! Cultivating such cells is also a process of cultivating patience!
Point 2: Grasp the inoculation density
Generally speaking, the density of suspension cells should not be lower than 500,000/ML. Many suspension cells are density-dependent, and non-proliferation caused by low density is also one of the common problems for novices. Of course, there are very few cells whose state becomes worse when the density is high, such as W6/32 (mouse B cell hybridoma cells). Therefore, it is very important to search for relevant information to grasp the characteristics of cells before cultivating unknown cells. Only by knowing yourself and the enemy can you win all battles!
The frequency of medium replacement should not be limited to "clearly". Cells are living things. During a continuous culture cycle, specific nutrients such as supplementary medium and glucose can be added several times depending on the growth status of the cells to maintain a good growth status of the cells. . During the suspension culture process, with the proliferation of cells, the consumption of nutrients in the original medium and the increase of metabolites, the viability of cells will decrease, and timely supplement of nutrients will make the cells in a relatively good living environment. I won’t go into details here, and avoid frequent centrifugation and liquid replacement.
Let's talk about the liquid replacement method:
①Centrifugal full liquid exchange method
That is, remove all the old medium by centrifugation (800-1000rpm, 5min), and replace with fresh medium. This method is suitable for medium replacement of "skin solid" well-raised suspension cells (such as K562), or the current situation where the cells are in good condition and the density is high, resulting in a large consumption of medium. The advantage of this method is that the medium is changed thoroughly and some cell debris can be removed, but it will also cause a certain degree of mechanical damage to the cells.
②Centrifugal half exchange method
That is, by centrifuging (800-1000rpm, 5min) 50% cell suspension, replace 50% volume of fresh medium. This method is suitable for comparing "hypocritical" difficult-to-culture cells (such as thp-1), or the situation where the density is low but more debris needs to be removed in the adjustment state.
③Sedimentation liquid exchange method
That is, instead of using a centrifuge, the method of natural sedimentation by gravity is used to separate the medium from the cells, so as to replace all or part of the fresh medium. However, it has application limitations—it is only suitable for cells that can form cell clusters of a certain size (otherwise they cannot settle naturally and can only be centrifuged at low speed), especially when dealing with cells that rely on cell aggregation to proliferate, such as NK-92, NK- 92 MI, Jurkat. This method can minimize the mechanical damage caused by the centrifugation process during the liquid exchange process, while retaining the aggregated cell mass, and the removal of cell debris is relatively thorough.
After mastering the above points, plus a good cell seed, it is just around the corner to grow "beautiful" suspension cells!
cell culture plate
Yongyue Medical's U-shaped cell culture plate is mostly used for culturing suspension cells, using medical grade polystyrene (PS), produced in a 100,000-level purification workshop, sterilized by irradiation, free of DNase, RNase, and pyrogen-free , Safety and environmental protection.
Cell culture plates are available in various specifications to meet the needs of various bacterial cultures. The product types are divided into 6-well, 12-well, 24-well, 48-well, 96-well, and 384-well, of which 96-well is divided into flat-bottom, U-bottom, V-bottom bottom; U-shaped culture plates are mostly used for culturing suspension cells; V-shaped culture plates are mostly used for immunological hemagglutination experiments.
