Characterization and application scenarios of various enzymes in the preparation of tissue single-cell suspensions

Tissue consists of cells and extracellular matrix (ECM) ordered and tightly linked together. Obtaining single-cell suspensions from tissues for applications such as flow assays, single-cell sequencing, in vitro cultures, drug screening, cell transplantation, organoid cultures, and tumor research is closer to the natural state in vivo and offers many advantages.

To obtain high-quality single-cell suspensions, some effective measures are needed, among which, enzymatic digestion is a method that can maintain the optimal state of cells while effectively obtaining individual cells.

There are various enzymes for digesting tissues, and the common digestive enzymes are mainly as follows. Depending on the principle of digestion, their application scenarios vary:

categorization	principle	appliance	caveat
trypsin	Acts on lysine- or arginine-conjugated peptide tendons to remove intercellular mucins and glycoproteins, affecting the cytoskeleton and thereby separating cells	For digesting soft tissues with little interstitial cell mass, such as embryonic, epithelial, liver and kidney tissues; not for fibrous tissues and harder cancerous tissues	EDTA can be used to enhance the hydrolysis of trypsin, and serum affects trypsin activity
Collagenase Ⅰ	Hydrolyzes interstitial proline, separates cells, digests only the interstitium, little damage to cells	Digestive epithelium, lung, fat, adrenal epithelium	Calcium and magnesium ions and serum do not affect collagenase activity and digestion
Collagenase II	Hydrolyzes interstitial proline, separates cells, digests only the interstitium, little damage to cells	Digestion of liver, bone, cartilage, tumor, thyroid, heart, salivary gland tissue cells	Calcium and magnesium ions and serum do not affect collagenase activity and digestion
Collagenase III	Hydrolyzes interstitial proline, separates cells, digests only the interstitium, little damage to cells	Digests all mammalian tissues	Calcium and magnesium ions and serum do not affect collagenase activity and digestion
Collagenase IV	Hydrolyzes interstitial proline, separates cells, digests only the interstitium, little damage to cells	Digests a wide range of tissues	Calcium and magnesium ions and serum do not affect collagenase activity and digestion
Collagenase V	Hydrolyzes interstitial proline, separates cells, digests only the interstitium, little damage to cells	Digestion of pancreatic islet tissue	Calcium and magnesium ions and serum do not affect collagenase activity and digestion
Hyaluronidase	Reduction of hyaluronic acid activity by stoichiometric degradation of the β-N-acetylhexosamine-[1-4] glycosidic bond of hyaluronic acid	Often paired with collagenase to digest connective tissue	Routinely stable and does not decompose
DNaseⅠ	Acts on DNA degraded by cell separation, avoiding DNA-induced cell agglutination, without damaging cellular integrity	Often used in conjunction with collagenase or hyaluronidase, etc., not usually used alone	Metal ion chelators such as EDTA, zinc ions up to mmol/L concentration, 0.1% SDS, reducing agents such as DTT and mercaptoethanol, and salt concentrations of 50-100 mM or more all showed significant inhibition of DNase Ⅰ.
Neutral protease	Neutral proteases only hydrolyze peptide bonds with amino groups provided by hydrophobic macromolecular amino acids such as leucine, phenylalanine, and tyrosine	Mild protease hydrolysis activity, will not damage the integrity of the cell membrane, often used in conjunction with collagenase, etc., its separation of fibrous tissue is more efficient than the epithelial tissue	Neutral proteases of most microorganisms are metalloenzymes, which are thermally unstable and easily autolysed. Calcium ions can increase the stability of the enzyme and reduce enzyme autolysis
Elastase	Hydrolyzes elastin by acting on its peptide, amide and ester bonds, hydrolyzes natural elastin	Often used in combination with trypsin and collagenase, etc. for isolation of connective tissues and tissues containing large amounts of cellular reticular fibers. Good for digesting lungs and isolating alveolar type II cells	Optimum pH 7.8, optimum temperature 25℃.
Papain	A mercapto protease that hydrolyzes the carboxyl terminus of arginine and lysine in proteins and peptides, and preferentially hydrolyzes those amino acids with two carboxyl groups at the N-terminus of the peptide bond or the peptide bond of aromatic L-amino acids	Commonly used to isolate cortical neurons, retina, smooth muscle	Optimum pH 6-7; optimum temperature 55-65°C, heat-resistant, not completely inactivated at 90°C; inhibited by oxidizing agents, activated by reducing substances

Due to the different cells and structures of different tissues, good results cannot be achieved using a single enzyme to digest the cells, and it is often necessary to mix and match several different enzymes to obtain the best performing single cell suspension. For specific tissues, experimental mapping is still required to determine the optimal digestion protocol. The following list of methods is for your reference.

Table 1: Reference to commonly used digestion protocols for human tissues

Form	Organization	Digestive enzyme	Reference PMID
Human - normal tissue	Myocardial tissue	Collagenase II (200 IU/mL)	28202059
Human - normal tissue	Liver disease	Collagenase D (2.5 mg/mL)	25533785
Human - normal tissue	Liver disease	DNaseⅠ（0.1 mg/mL）	25533785
Human - normal tissue	Lung parenchyma	Collagenase (300 U/mL)	3026698
Human - normal tissue	Lung parenchyma	DNaseⅠ（50 U/mL）	3026698
Human - normal tissue	Lungs	Protease XIV (2 mg/mL)	7681268
Human - normal tissue	Lungs	Lactase (0.5 mg/mL)	7681268
Human - normal tissue	Gallbladder	Collagenase I (0.025%)	31385550
Human - normal tissue	Tooth pulp	Collagenase I (2 mg/mL)	24831838
Human - normal tissue	Tonsil	Collagenase (1 mg/mL)	27598832
Human - normal tissue	Tonsil	DNaseⅠ（0.25 mg/mL）	27598832
Human - normal tissue	Placental tissue	Collagenase II (0.25%)	30122114
Human - normal tissue	Placental tissue	Trypsin (0.25%)	30122114
Human - normal tissue	Afterbirth	Trypsin (0.25%)	30541665
Human - Tumor tissue	Squamous cell carcinoma	Trypsin (0.05%)	2452013
Human - Tumor tissue	Squamous cell carcinoma	EDTA（2 mM）	2452013
Human - Tumor tissue	Neurofibroma	Neutral protease (1.25 U/mL)	1696266
Human - Tumor tissue	Neurofibroma	Collagenase I (0.05%)	1696266
Human - Tumor tissue	Neurofibroma	Hyaluronidase (0.1%)	1696266
Human - Tumor tissue	Glioma	Collagenase II, IV, V, Ⅺ (1 mg/mL)	27598832
Human - Tumor tissue	Glioma	DNaseⅠ（0.25 mg /mL）	27598832
Human - Tumor tissue	Melanoma (type of skin cancer)	Collagenase IV (1 mg/mL)	23525090
Human - Tumor tissue	Melanoma (type of skin cancer)	DNaseⅠ（0.1 mg/mL）	23525090
Human - Tumor tissue	Lung tumor	Collagenase I (170 mg/L)	25359999
Human - Tumor tissue	Lung tumor	Collagenase II (56 mg/L)	25359999
Human - Tumor tissue	Lung tumor	Collagenase IV (170 mg/L)	25359999
Human - Tumor tissue	Lung tumor	DNaseⅠ（0.002%）	25359999
Human - Tumor tissue	Lung tumor	Elastase (0.002%)	25359999
Human - Tumor tissue	Lymphoma	Collagenase I (4 mg/mL)	30692706
Human - Tumor tissue	Lymphoma	Hyaluronidase (1 mg/mL)	30692706
Human - Tumor tissue	Lymphoma	Trypsin (0.1%)	30692706
Human - Tumor tissue	Colon	Trypsin (0.25%)	6830688

表二：小鼠组织常用的消化方案参考

Category	Tissue	Digestive Enzymes	Reference PMID
Mouse - normal tissue	Respiratory	Collagenase IV (0.1%)	17577582
Mouse - normal tissue	Respiratory	Trypsin (0.2%)	17577582
Mouse - normal tissue	Lungs	Elastase (4 U/mL)	29956144
Mouse - normal tissue	Lungs	Neutral protease (1 U/mL)	29956144
Mouse - normal tissue	Lungs	DNaseⅠ（200 μg/mL）	29956144
Mouse - normal tissue	Thymus gland	Collagenase D (0.125%)	25367128
Mouse - normal tissue	Thymus gland	DNaseⅠ（0.1%）	25367128
Mouse - normal tissue	Gastric	Neutral protease II (0.72 mg/mL)	29322413
Mouse - normal tissue	Gastric	Collagenase A (1 mg/mL)	29322413
Mouse - Tumor Tissue	Lung cancer	Neutral protease (50 U/mL)	22064658
Mouse - Tumor Tissue	Lung cancer	Collagenase (400 U/mL)	22064658
Mouse - Tumor Tissue	Lung cancer	DNaseⅠ（50 U/mL）	22064658
Mouse - Tumor Tissue	Breast tumor	Collagenase (1.5 mg/mL)	23959163
Mouse - Tumor Tissue	Breast tumor	Hyaluronidase (20 μg /mL)	23959163

Table 3: Reference to commonly used digestion protocols for rat tissues

Category	Tissue	Digestive Enzymes	Reference PMID
Rat - normal tissue	Respiratory	Collagenase II (200 IU/mL)	28202059
Rat - normal tissue	Liver disease	Collagenase IV (0.5 mg/mL)	27054325
Rat - normal tissue	Liver disease	DNaseⅠ（20 μg/mL）	27054325
Rat - normal tissue	Gallbladder	Collagenase (1 mg/mL)	28668953
Rat - Tumor Tissue	Colon	Collagenase I (1.6 mg/mL)	23193035
Rat - Tumor Tissue	Colon	Hyaluronidase (20 μg /mL)	23193035
Rat - Tumor Tissue	Colon	DNaseⅠ（60 μg/mL）	23193035

Example

The enzyme digestion process and staining procedure of 4T1 hormonal mice are shown below:

(1) Preparation of 10× mixed digestive enzymes: take 80 mL of HBSS buffer, add 1 g of collagenase IV, 100 mg of hyaluronidase, to reduce cell viscosity and agglutination, add an additional 20,000 U of DNase Ⅰ, fully dissolve and mix, replenish HBSS buffer, and then fix it to 100 mL, filter it using 0.22 μm filter membrane, dispense it into 5 mL/strip, and store it at -20℃ and return to room temperature before use;

(2) Tumor isolation: The tumor-bearing mice were executed by cervical dislocation method, placed in a beaker containing 75% alcohol, immersed for 3~5 min, stripped of tumors using scissors and forceps, and washed twice with PBS;

(3) Digestion of tumor tissue: transfer the tumor to a 1.5 mL EP tube, hold the curved scissors, insert the EP tube and repeatedly shear to a pellet of about 0.5~1 mm3 in size, make up 1 mL of HBSS buffer, transfer it to a 15 mL centrifuge tube, and then resuspend the tumor pellet using 8 mL of HBSS buffer, add 1 mL of 10×mixed digestive enzymes, gently blow and mix, and then digest the pellet on a shaking table at 80 rpm, 37℃ for 1~2 h;

★Note: 10 mL of digestive solution can adequately digest tumor tissue with a volume of about 1 cm3.

(4) Preparation of single-cell suspension: use a 1 mL pipette to blow and aspirate the tissue block every 30 min, until there is no obvious obstruction to the blowing and the digestion is considered to be complete, remove the impurities by filtering through a 100 μm sieve, and use a large volume of about 50 mL of HBSS buffer to wash once, centrifuge at 300 g for 5 min, discard the supernatant, and then resuspend the cellular precipitate by adding an appropriate volume of HBSS buffer or PBS buffer, i.e. tumor single-cell suspension. The cell precipitate should be resuspended with appropriate volume of HBSS buffer or PBS buffer, which is the single cell suspension of the tumor;

(5) Flow staining: cells were counted, adjusted to the appropriate density (1*107/mL), and flow stained and analyzed.

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