Enzyme Selection for Single-Cell Suspension Preparation

Enzymatic digestion is one of the commonly used methods for preparing single-cell suspensions. It utilizes specific enzymes to carry out corresponding chemical reactions to remove certain specific structures, making it suitable for digesting small tissue blocks into single cells. Collagenase is an important enzyme species for single-cell tissue digestion, and collagenase digestion is an indispensable step in the preparation of single-cell samples. In addition, common digestive enzymes for tissue dissociation include Trypsin, Deoxyribonuclease, Hyaluronidase, Papain, Neutral Protease, and Elastase. Different enzymes are suitable for different tissue types.

1. Collagenase

Collagenase mainly hydrolyzes collagen components in connective tissues. Currently, commercial collagenase is extracted and prepared from Clostridium histolyticum, and is classified into different types such as Type Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ. The type of collagenase should be selected according to the type of tissue to be separated and digested.

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2. Trypsin

Trypsin is an endopeptidase that selectively hydrolyzes arginine and lysine peptide bonds, and can hydrolyze natural proteins, denatured proteins, fibrin, and mucin into polypeptides or amino acids. Trypsin can be applied to the digestion of liver, kidney, thyroid, and other tissues, and needs to be used together with other proteases such as collagenase or elastase.

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3. Deoxyribonuclease Ⅰ (DNase Ⅰ)

Deoxyribonuclease Ⅰ (DNase Ⅰ) is generally used in the process of enzymatic cell separation experiments. DNase Ⅰ does not damage cell integrity. It digests DNA released into the solution due to cell damage, thereby increasing viscosity and viability.

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4. Hyaluronidase (HAase)

Hyaluronidase (HAase) is a general term for enzymes that can hydrolyze hyaluronic acid. It is an enzyme that can reduce the activity of hyaluronic acid in the body, thereby improving the fluid permeability in tissues. It is often combined with natural proteases such as collagenase for dissociating connective tissues.

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5. Papain

Papain is used in cell culture and is used to separate cells in the first step of preparing cell culture medium. After treating small tissue blocks with the enzyme for 10 minutes, the extracellular matrix connecting cells can be broken; then protease inhibitors are used to stop the reaction to prevent papain from further lysing the cells themselves. Compared with trypsin and elastase, papain can decompose most protein substrates and is often used to dissociate cortical neurons, retina, smooth muscle, and other tissues.

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6. Neutral Protease (Dispase)

Neutral Protease (Dispase), also known as dispase, is derived from Bacillus subtilis and is free of mycoplasma or other animal virus contamination. It has relatively mild proteolytic enzyme activity, causes little damage to cells, and can maintain cell membrane integrity, so it is very suitable for the isolation of primary and subcultured cells. Neutral protease can also be used to eliminate cell aggregation that occurs during suspension cell culture. It is often used in combination with collagenase or other proteases.

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7. Elastase

Elastase is a serine protease derived from porcine pancreas that catalyzes the hydrolysis of proteins and polypeptides (especially peptide bonds next to neutral amino acid residues). Among all proteases, elastase is very unique because it can hydrolyze natural elastin. Elastase has been found in blood and bacteria. Studies have found that elastase is very suitable for digesting lungs to isolate type Ⅱ alveolar cells.

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Brand Mission

ANT BIO PTE. LTD. is dedicated to advancing life science research by providing high-quality, reliable reagents and comprehensive solutions. We recognize the critical challenges in single-cell suspension preparation and the urgent need for standardized, specialized digestive enzymes. Through our specialized sub-brands (Absin, Starter, UA), we have developed a targeted product portfolio for tissue dissociation and single-cell research, covering core digestive enzymes such as collagenase, trypsin, deoxyribonuclease Ⅰ, and other supporting proteases.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the high purity, stability, and biological activity of each enzyme product. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as tissue-specific digestion efficiency, cell viability maintenance, and enzyme compatibility, and accelerating the pace of scientific research breakthroughs in single-cell biology and related fields. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of single-cell research technology and the development of innovative biological research methods.

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This article is AI-compiled and interpreted based on the original work related to tissue digestive enzyme research. All intellectual property (e.g., experimental protocols, data, product information) of the original publication shall belong to the relevant research team. For any infringement, please contact us promptly and we will take immediate action.

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At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of tissue dissociation and single-cell research needs, from core digestive enzymes to related supporting products. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in overcoming single-cell suspension preparation challenges and driving progress in single-cell biology research. Explore our product portfolio today and elevate your research to new heights.