Tumor Tissue Dissociation Kit, IC-1120
Product Introduction
This kit provides a standardized enzymatic digestion system for the gentle and efficient dissociation of human or mouse solid tumor tissues into single-cell suspensions or small cell clusters, suitable for downstream flow cytometric sorting, primary cell culture, and organoid construction. The core enzymatic digestion system consists of Collagenase IV, Collagenase I, DNase I, and Hyaluronidase, dissolved in Hanks'''' balanced salt solution containing calcium and magnesium ions, balancing matrix degradation efficiency with preservation of cell surface markers. In this version, all enzyme components are provided as lyophilized powders; users must reconstitute, filter-sterilize, aliquot, and prepare working solutions themselves. The amount per vial far exceeds the volume required for a single experiment; repeated freeze-thaw cycles after reconstituting an entire vial are strictly prohibited. This product is for research use only and is not intended for clinical diagnosis or treatment.
Product Features
1. Standardized Multi-Enzyme System: Collagenase IV, Collagenase I, DNase I, and Hyaluronidase work synergistically for efficient tissue dissociation.
2. Cell Surface Marker Preservation: Balanced enzyme composition minimizes damage to cell surface receptors, supporting downstream flow cytometry applications.
3. Multi-Application Compatibility: Suitable for flow cytometric sorting, primary cell culture, and organoid construction.
4. Lyophilized Powder Format: Enzymes are provided as lyophilized powders for extended shelf life and flexible reconstitution.
5. Two Standard and Enhanced Modes: Standard mode for flow sorting and primary culture; enhanced mode for fibrous stroma-rich tumors or organoid initiation.
Specifications
Size: 100 mL (25 isolations)
Components:
Component A1: Collagenase IV lyophilized powder, 100 mg × 1 vial, store at 2-8°C, dry and protected from light
Component A2: Collagenase I lyophilized powder, 100 mg × 1 vial, store at 2-8°C, dry and protected from light
Component B: DNase I lyophilized powder, 25 mg × 1 vial, store at 2-8°C, dry
Component C: Hyaluronidase lyophilized powder, 100 mg × 1 vial, store at -20°C, dry
Component D: Digestion Buffer (Hanks'''' with Ca²⁺/Mg²⁺), 100 mL × 1 bottle, store at 2-8°C
Buffer D Formulation: Hanks'''' balanced salt solution containing calcium and magnesium ions, supplemented with 0.5% (w/v) BSA, 100 U/mL penicillin, and 100 µg/mL streptomycin
Storage and Stability
Storage Conditions (Unreconstituted): Store according to the conditions listed for each component. Shelf life is 12 months, based on the expiration date on the label. Component C (Hyaluronidase) is moisture-sensitive; store at -20°C and equilibrate to room temperature before opening to prevent condensation.
Storage Conditions (Reconstituted Stocks):
- Collagenase A1 and A2 stocks: store at -20°C protected from light, use within 7 days
- DNase I stock B: store at -20°C, use within 1 month
- Hyaluronidase stock C: store at -20°C, use within 3 months
All stocks must not undergo more than 2 freeze-thaw cycles. Working solutions: prepare fresh and use within 2 hours.
Applicable Sample Types
Human solid tumor surgical specimens and biopsy tissues.
Mouse xenograft tumors and spontaneous tumor model tissues.
Applicable tumor types: breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, etc. For specimens with extremely rich fibrous stroma such as pancreatic cancer, extended digestion time or enhanced mode is recommended.
Not applicable: blood samples, bone marrow aspirate, established cell cultures.
Principle
The extracellular matrix of tumor tissue is mainly composed of collagen, hyaluronic acid, and proteoglycans. This kit achieves dissociation through multi-enzyme synergy. Collagenase IV preferentially degrades basement membrane type IV collagen, causing less damage to epithelial and endothelial cells, which helps preserve cell surface receptors. Collagenase I acts on interstitial type I and type III collagen, targeting tumor fibrous septa and stromal matrix. Hyaluronidase hydrolyzes hyaluronic acid, reducing matrix viscosity and enhancing enzyme penetration into dense stroma. DNase I degrades viscous DNA released from dead cells, preventing cell aggregation and improving single-cell suspension quality.
Isolation Count Description
The nominal isolation count is based on approximately 1 g of tissue using 4-5 mL of standard working solution. Collagenase IV content is 100 mg, sufficient to prepare 100 mL of standard working solution with a final Collagenase IV concentration of 1 mg/mL, giving 25 isolations at 4 mL each or 20 isolations at 5 mL each conservatively; Buffer D provided is 100 mL. Note: Collagenase IV is the bottleneck component and determines the maximum working solution volume; Collagenase I, DNase I, and Hyaluronidase are provided in excess. The nominal count uses a compact ratio of 4 mL per gram of tissue, suitable for soft tissue tumors with less stroma; fibrous stroma-rich tumors such as pancreatic and breast cancer often require 5-10 mL per gram of tissue or enhanced mode, with actual counts reduced to approximately 20. Buffer D volume is strictly matched to the Collagenase IV-limited working solution volume.
Protocol
I. Batch Reconstitution Protocol (Critical Step, Perform Under Aseptic Conditions)
Why batch reconstitution: Reconstituting an entire 100 mg vial requires 10 mL of solvent, resulting in many aliquots and difficulty controlling contamination and freeze-thaw risks. Batch reconstitution according to experimental frequency is recommended: reconstitute only the required amount of powder each time and keep the remaining powder dry and sealed. Recommended dry powder aliquoting: in a biosafety cabinet, divide the powder into several portions using a sterile spatula, e.g., 10 mg × 10 portions or 20 mg × 5 portions, into sterile, dry, capped centrifuge tubes, seal, and store under unreconstituted conditions. Avoid moisture absorption during weighing; work quickly and keep caps closed. If weighing is not feasible, whole-vial reconstitution followed by immediate single-use aliquoting may be used, but is recommended only for Collagenase A1 and A2, and must be aliquoted and frozen immediately after reconstitution.
II. Enzyme Stock Reconstitution (Example: 10 mg Dry Powder per Single Use)
General Principle: Use sterile water for injection or Buffer D without enzyme for reconstitution. Gently vortex until completely dissolved; avoid vigorous stirring that generates bubbles, as bubbles cause enzyme protein denaturation. After reconstitution, filter-sterilize through a 0.22 µm low-protein-binding filter.
A1 Collagenase IV: Single amount 10 mg, solvent volume 1.0 mL, stock concentration 10 mg/mL, 0.22 µm filtration, aliquot 100 µL per tube, store at -20°C protected from light, use within 7 days.
A2 Collagenase I: Single amount 10 mg, solvent volume 1.0 mL, stock concentration 10 mg/mL, 0.22 µm filtration, aliquot 100 µL per tube, store at -20°C protected from light, use within 7 days.
B DNase I: Single amount 5 mg, solvent volume 0.5 mL, stock concentration 10 mg/mL, 0.22 µm filtration, aliquot 100 µL per tube, store at -20°C, use within 1 month.
C Hyaluronidase: Single amount 10 mg, solvent volume 10.0 mL, stock concentration 1 mg/mL, 0.22 µm filtration, aliquot 1 mL per tube, store at -20°C, use within 3 months.
Note: Specification A DNase I is 25 mg per vial; divide into 5 portions of 5 mg each and reconstitute 5 mg in 0.5 mL at 10 mg/mL per use. Specification B 50 mg may be divided into 10 portions of 5 mg each. Hyaluronidase is reconstituted at 1 mg/mL rather than 10 mg/mL because high concentrations are viscous and difficult to filter, and the working solution final concentration requires only 0.1 mg/mL. Do not vortex DNase I; gently pipette or invert until dissolved to prevent inactivation by shear stress. If the label indicates activity units, calculate reconstitution volume by activity to achieve equivalent stock activity. Post-reconstitution quality control: the solution should be clear or slightly yellow; if obviously turbid or containing precipitate, discard and reconstitute again.
III. Working Solution Preparation (Immediately Before Use, Under Aseptic Conditions)
Prepare standard working solution according to the following ratios, using 10 mL working solution as an example. A1 Collagenase IV stock 10 mg/mL, add 1.0 mL, final concentration 1 mg/mL. A2 Collagenase I stock 10 mg/mL, add 0.5 mL, final concentration 0.5 mg/mL. B DNase I stock 10 mg/mL, add 0.1 mL, final concentration 0.1 mg/mL. C Hyaluronidase stock 1 mg/mL, add 1.0 mL, final concentration 0.1 mg/mL. D Buffer at 1× concentration, bring to 10 mL, final concentration 1×. Mode selection: Standard mode for flow sorting and primary culture, digest 30-45 minutes; enhanced mode for fibrous stroma-rich tumors or organoid initiation, A2 increased to 1.0 mL (final 1 mg/mL), C increased to 1.5 mL (final 0.15 mg/mL), digest 45-60 minutes. Mix and pre-warm in a 37°C water bath for 5 minutes; use within 2 hours.
IV. Tissue Pretreatment
Place fresh tumor tissue in pre-chilled antibiotic-containing buffer (enzyme-free Component D may be used) and complete processing within 4 hours at 4°C. In a sterile Petri dish, remove necrotic tissue, fat, and obvious fibrous tissue, and mince the tissue into 1-2 mm³ pieces with sterile scissors. Wash the tissue pieces twice with enzyme-free Component D and discard the supernatant.
V. Enzymatic Digestion
Resuspend tissue pieces in pre-warmed working solution at a ratio of 4-10 mL per gram of tissue. Place in a 37°C shaker or water bath with low-speed oscillation at 80-100 rpm. Every 10-15 minutes, gently pipette 3-5 times, avoiding bubbles. Digestion endpoint: every 15 minutes, take 10 µL of digestion solution for microscopic examination. When numerous dispersed single cells are visible, tissue pieces have largely disappeared, and trypan blue viability is ≥85%, terminate immediately. Standard mode total time should not exceed 45 minutes; enhanced mode should not exceed 60 minutes. Warning: Digestion beyond 60 minutes will significantly increase cell death and the risk of signal loss for certain surface markers such as CD4, CD8, CD45RA, and CD14.
VI. Termination of Digestion and Filtration
Add at least an equal volume of complete medium containing 10% fetal bovine serum, or buffer containing 2 mM EDTA, to terminate digestion, and mix gently. Filter the suspension through a 70 µm cell strainer into a centrifuge tube. For flow sorting applications, filter a second time through a 40 µm strainer. Centrifuge at 300 x g for 5 minutes at 4°C and discard the supernatant. Resuspend the cell pellet in an appropriate buffer: for flow sorting, use PBS containing 0.5% (w/v) BSA and 1 mM EDTA, keep on ice; for primary culture, use the appropriate basal medium, preferably serum-free, to avoid residual enzyme affecting attachment; for organoid culture, use serum-free basal medium before mixing with Matrigel.
Downstream Application Guide
I. Flow Cytometric Sorting
Digestion time control: Standard mode with 30-minute digestion is recommended. Literature clearly shows that collagenase plus DNase digestion can reduce CD4, CD8, CD45RA, and CD14 flow signals; hyaluronidase-containing formulations may reduce CD54 (ICAM-1) by approximately 30%. If targets include these markers, set undigested controls in data analysis or use a conservative protocol with shorter digestion time. Before analysis, filtration through a 40 µm strainer is mandatory to prevent nozzle clogging. Sorting buffer recommendation: PBS plus 0.5% (w/v) BSA plus 1 mM EDTA, operate on ice throughout.
II. Primary Cell Culture
After terminating digestion, wash cells twice more with enzyme-free buffer to thoroughly remove residual collagenase; residual enzyme will digest culture dish coating matrix and affect attachment. Seeding viability threshold should be ≥90%, determined by trypan blue or acridine orange/propidium iodide counting. Below this threshold, DNA and proteases released from dead cells will impair primary cell attachment and proliferation. DNase I is particularly critical in primary culture: viscous DNA released from dead cells is the main cause of cell clumping and uneven seeding.
III. Organoid Culture
Digestion endpoint: digest to small, dense cell clusters rather than completely single cells. After 70 µm filtration, small clusters retained on a 40 µm strainer can be used for organoid seeding. Matrigel compatibility: residual serum proteins after terminating digestion may interfere with Matrigel polymerization; wash once with serum-free basal medium before mixing with Matrigel. For organoid initiation from primary tumor tissue, the collagenase, DNase I, and hyaluronidase system is gentler than single-enzyme systems such as TrypLE and better preserves stem cell activity.
Quality Control
Collagenase Activity A1 and A2: Method: Wünsch collagenase activity assay; acceptance criterion: ≥90% of labeled value.
DNase Activity B: Method: DNA agarose gel electrophoresis or spectrophotometry; acceptance criterion: ≥1 µg lambda DNA degraded per µL stock within 5 minutes after reconstitution.
Hyaluronidase Activity C: Method: turbidimetric or equivalent method; acceptance criterion: ≥90% of labeled value.
Fill Volume Variation: Method: gravimetric; acceptance criterion: 95-105% of labeled amount.
Sterility (Lyophilized Powder): Method: membrane filtration; acceptance criterion: no growth.
Endotoxin: Method: LAL assay; acceptance criterion: ≤1 EU/mL at working concentration.
Post-Reconstitution Functional Validation: Method: reconstitute according to instructions and digest standard tumor cell line clusters or mouse xenograft tumors; acceptance criterion: viability ≥85% and single-cell yield ≥1 million cells per gram of tissue.
Precautions
For research use only; not for clinical diagnosis, treatment, food, or drug use. Fill volume far exceeds single-use amount; repeated freeze-thaw cycles after reconstituting an entire vial are strictly prohibited. Batch reconstitution with dry powder aliquoting or immediate single-use aliquoting after whole-vial reconstitution is recommended; more than 2 freeze-thaw cycles will significantly reduce enzyme activity. Reconstitution and filtration are critical steps: lyophilized powder must be completely dissolved before filtration; use low-protein-binding 0.22 µm filters, as ordinary filters may cause enzyme protein adsorption loss. If the solution is obviously turbid after filtration, reconstitute again. Hyaluronidase is reconstituted at 1 mg/mL rather than 10 mg/mL; avoid vortexing DNase I. Perform operations under aseptic conditions in a biosafety cabinet, wearing laboratory coat, gloves, and goggles. Collagenase I and hyaluronidase affect certain cell surface markers: for flow sorting applications, always refer to the warnings in the flow sorting section. Matrix density varies greatly among tumor types. If large tissue pieces remain after 60 minutes of digestion, add a small amount of working solution to continue digestion, but total time should not exceed 90 minutes; residual undigested tissue can be removed through a 100 µm strainer. Collagenases in this product are of bacterial origin, DNase I is of recombinant origin, and hyaluronidase is of bovine or ovine testicular origin. Operators allergic to these source proteins should exercise caution. Discard enzyme solutions and tissue waste according to laboratory biosafety regulations.
References
1. Frolova AA, Gerashchenko TS, Patysheva MR, et al. Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing. Bull Exp Biol Med. 2023;175(4):519-523. PMID: 37770788.
2. Howard RB, Shroyer KR, Marcell T, et al. Time-related effects of enzymatic disaggregation on model human lung carcinomas. Cytometry. 1995;19(2):146-153. PMID: 7743895.
3. Rong GH, Grimm EA, Sindelar WF. An enzymatic method for the consistent production of monodispersed viable cell suspensions from human solid tumors. J Surg Oncol. 1985;28(2):131-133. PMID: 2982062.
Ordering Information
Catalog Number: IC-1120
Product Name: Tumor Tissue Dissociation Kit
Size: 100 mL (25 isolations)
Suggested Retail Price: CNY ¥6050.00 / USD $605.00 / EUR �726.00 / JPY ¥108900.00
Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.
