Dissociation of cells with trypsin

Current research has identified a variety of protein hydrolases that can be used to dissociate cells, the most commonly used of which is trypsin, a member of the serine protease family. In the pancreas the precursor of trypsin is synthesised by trypsinogen and secreted as a component of the pancreatic juice. It is restricted by enterokinase, or trypsin, to become activated trypsin, a peptide chain endonuclease that cuts off the carboxyl side of the lysine and arginine residues in the peptide chain. Trypsin solutions are most potent at pH 7-9 and at 37°C. Prolonged incubation with high trypsin concentrations will strip the cell surface proteins and kill the cells.

EDTA (ethylenediaminetetraacetic acid): In the presence of calcium, adhesion molecules determine cell-cell and cell-matrix interactions. To attenuate this interaction, EDTA is often added to trypsin, which chelates divalent cations (Ca2+, Mg2+).

Phenol Red: The optimum activity of trypsin is achieved in the pH range of 7 to 9. Phenol Red in this range has a pinkish colour. Due to environmental conditions, the pH of trypsin may become acidic, orange in colour and make trypsin less effective. Trypsin activity can be optimised by adjusting the pH to 7.4-7.6 with NaOH.

Diluent: Concentrated trypsin is dissolved or diluted in a buffered salt solution that does not contain Ca2+ or Mg2+, such as Hank's Balanced Salt Solution (Product No. E301773), to maintain pH and osmotic balance. In addition, some trypsin products contain 0.9% NaCl as a diluent rather than Hank's Balanced Salt Solution.

Source and form: The main source of trypsin is the pig, which is supplied as a lyophilised powder form or as a solution. To avoid animal or microbial products, recombinant bovine trypsin is also expressed in maize, called Trypzean solution.

Concentration: Depending on the cell type and application, the concentration of trypsin needs to be adjusted. For strongly adherent cell lines, trypsin at 2.5% to 0.25%(1 x to 10 x times dilution) is used. Lower concentration (0.05% trypsin) solutions are generally used, although there are studies that require cell surface protein integrity.

Experimental programme

Pre-warm the trypsin solution, the balanced salt solution (Ca2+ and Mg2+ free solution) and the growth medium to 37 °C.

Check the cells to ensure they are healthy and free of contamination.

Remove from the culture flask and discard the medium.

Cells are gently washed with a balanced salt solution free of Ca2+ and Mg2+ ions and then the solution is removed. Cells firmly adherent to the wall can also be washed with trypsin solution. However, for sensitive cells, balanced salt solutions are preferred.

Add an appropriate amount (0.5mL/10cm2) of pre-warmed trypsin solution to the side wall of the culture flask. Gently swirl the contents to cover the cell layer.

Incubate the vessel at room temperature for 2-3 minutes. Firmly adherent cells can be detached quickly at 37°C. Observe the cells under the microscope. Detached cells appear round and refractive under the microscope. If less than 90% of the cells are detached, incubate the flask for a further 2 minutes and observe the cells under the microscope at 30 second intervals.

Note: Prevent cells from being exposed to trypsin solution for too long (≥ 10 minutes).

After the cells have been dislodged, add 2 times the volume of pre-warmed complete growth medium to inactivate the trypsin. Gently pipette the medium several times onto the surface of the cell layer to ensure cell recovery > 95%. For serum-free cultures, add Soya Trypsin Inhibitor (Product No.T113170) at equimolar concentrations to inhibit trypsin.

Note: Vigorous pipetting can lead to cell damage.

Transfer the cell suspension to a test tube and centrifuge gently at 300-1000×g for 5-10 minutes. Remove the supernatant and gently resuspend the cell pellet in pre-warmed complete growth medium. Remove the desired sample to determine the cell density of live cells using a blood cell counter and a Taipan Blue (product No. T293438) exclusion method or an automated cell counter.

The remaining solution was diluted to inoculum density and the appropriate volume pipetted into culture flasks and placed in the incubator.

Culture flask area	Volume of culture medium
25 cm2	5 – 10 mL
75 cm2	10 – 30 mL
175 cm2	40 – 150 mL

Common problems and solutions for cell dissociation

Cells are difficult to detach from the culture flask	·Loss of enzyme activity (trypsin) or low concentration	·Use the optimum concentration of fresh enzymes
	·Presence of serum (inhibition of trypsin activity)	·Thorough rinsing to remove serum
	·High degree of confluence and inaccessibility of enzymes to the cell-substrate interface	·Dislodging of cells at a lower cell density
Difficulty with cell adhesion	·High concentration of trypsin was used	·Trypsin digestion at optimum concentration and in less time
	·Incomplete inactivation of trypsin	·Addition of serum to inactivate trypsin
	·Inadequate attachment factors and serum	·Use antibiotics to rescue the culture and discard the batch of infected cells if the contamination has not been removed after 14 days.
	·Pollution
	·Citrate reduces the availability of calcium, thus reducing cell attachment.	·Remove and reduce the level of citrate used in cell culture systems.
	·Cell membrane damage	·Avoid prolonged exposure to trypsin and store the medium and cultures away from fluorescent lights to reduce the toxic effects of free radicals.
Detached and hypovitalised cells	·Pollution	·Discard contaminated cells
	·Lack of adequate attachment factors and serum	·Addition of appropriate adhesion factors and serum
	·High confluence of cells	·Dispersing and evacuating cells
	·Oxidative stress causes cells to round off and detach from substrates	·Minimising oxidative stress by adding albumin, glutathione
	·Change in osmolality (pH) of trypsin solution	·Checking the pH of the trypsin solution
Clots after cell shedding	·High concentration of trypsin was used	·Trypsin digestion at optimum concentration
	·Vigorous blowing and sucking	·Gentle blowing and sucking
	·Vigorous, prolonged centrifugation	· Gentle centrifugation (125 x g for 10 minutes)
	·Increased age of monolayer cells	·Dispersing and spreading cells before they reach 100% confluence
Cell membrane damage and cell death	·Increased lipid peroxidation, degrading the cell membrane leading to cell detachment	·Addition of ascorbic acid and glutathione to inhibit lipid peroxidation
	·Lack of plasma copper cyanide in serum-free medium catalyzes the formation of hydroxyl radicals and damages cell membranes.	·Add albumin and glutathione to the formulation to reduce oxidative damage
	·The concentration of iron in the solution increases and it supports free radical activity on the cell surface, damaging the cell membrane.	·Isolated iron delivery to cells by transfer
	·Hydrogen peroxide forms hydroxyl radicals in the presence of ferrous and cuprous ions	·Mannitol/pyruvate was added to the medium to complex and stabilise the hydrogen peroxide.
	·Riboflavin photolysis to form hydrogen peroxide	· Adding fresh riboflavin to cell cultures to avoid prolonged exposure of the cell culture system to light
	·In the presence of oxides, peroxides, carbonates, hydroxyl, phosphate and sulphide anions, precipitation results in the loss of zinc.	·Add zinc in complex with albumin/insulin to increase zinc content and minimise oxidative stress to prevent formation of oxides, peroxides and sulphides.