Collagenase Type III targets collagen types 1, 4, 9, 10, and 14, as well as fibronectin, MMP-9, gelatin, plasminogen, aggrecan, and perlecan osteonectin. It is a member of the matrix metalloproteinase (MMP) family and functions as a zinc peptidase.

Collagenase, Type III is used for the preparation of primary breast cells.
Preparation of stock solution
1. Add 1 mL of HBSS (Hank equilibrium salt solution, containing Ca2+and Mg2+) to 100 mg of collagenase, shake to fully dissolve, and prepare a storage solution of 100 mg/mL (i.e. 100 x);
2. Use low protein binding 0.22 μ M filter membrane filtration for sterilization, immediately use or divide into small portions, and freeze in the dark at -20 ° C to -5 ° C;
3. Thaw on ice before use to avoid repeated freeze-thaw cycles. The commonly used concentration for tissue and cell dispersion is 0.5-2.5 mg/mL, and the commonly used concentration for cartilage digestion is 1-2mg/mL. The optimal working concentration required needs to be determined based on specific experimental conditions or reference to relevant literature.
Separation of Organ/Tissues
1. Use sterile surgical knives or scissors to cut tissue into 3-4 mm sized tissue blocks;
2. Wash tissue blocks several times using HBSS containing Ca2+and Mg2+;
3. Add sufficient HBSS containing Ca2+and Mg2+to immerse the tissue block, and add collagenase to the required working concentration;
4. Incubate at 37 ° C for 4-18 hours. Using a horizontal shaker and supplementing digestion with 3 mM CaCl2 during digestion can improve digestion efficiency;
5. The dispersed cells can be screened using stainless steel or nylon mesh, and collected for later use. Add an appropriate amount of fresh collagenase working solution to the partially dissociated tissue and continue incubation at 37 ° C for further digestion;
6. Wash the collected cells several times using HBSS without collagenase;
7. Resuspend the above cells in cell culture medium and calculate the density of live cells using an automatic cell counter or other methods;
8. Inoculate cells using appropriate cell culture medium on a cell culture dish.
Organ perfusion
1. Adding collagenase to HBSS containing Ca2+and Mg2+preheated at 37 ° C, and adding 3 mM of CaCl2 can help improve separation efficiency;
2. Inject collagenase working solution into the corresponding organs at the optimized rate;
3. The recovered infusion solution from the above process is passed through a stainless steel or nylon mesh screen to separate the dissociated cells or small fragments of tissue from larger clumps. The tissue that has not been fully decomposed needs to be further incubated with fresh collagenase working solution at 37 ° C.
4. Same as steps 6-8 in the above section "Separation of Organ/Tissues".

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In vitro, Collagenase Type III is used for the dissociation of mammary glands, colon cancer stem cells, and breast epithelial tumor cells. It exhibits low levels of proteolytic activity, especially for caseinase, clostripain, and trypsin, making it suitable for applications requiring minimal damage to non-target proteins.

In vivo, collagenase enzymes in general have been shown to have therapeutic effects in wound healing, burns, and various diseases. The specific in vivo applications of Type III collagenase are less documented, but its use in research for tissue dissociation is well established.

In vitro enzyme assays for Collagenase Type III measure its collagenolytic activity. One unit is defined as the amount of enzyme required to hydrolyze collagen to produce 1 μmol of L-Leucine in 5 hours at 37°C, pH 7.5. The enzyme's activity is optimal at pH 6.3-8.5.

In vitro cellular assays for Collagenase Type III involve its use in tissue dissociation. Tissues are incubated with the enzyme in HBSS containing Ca²⁺ and Mg²⁺ at 37°C for 4-18 hours. The resulting cell suspension is filtered, washed, and used for primary cell culture. The enzyme is also used in organ perfusion protocols.

In vivo animal studies for Collagenase Type III are not extensively documented. However, collagenases in general have been studied in animal models for their effects on tissue dissociation and cell isolation. The enzyme's low secondary protease activity makes it suitable for specific applications where preservation of cell surface proteins is important.

Pharmacokinetic data for Collagenase Type III are not typically reported, as it is a research reagent. When used in tissue dissociation, the enzyme is removed after digestion. For therapeutic applications, the enzyme is administered locally and acts at the site of application.

Collagenase Type III has low systemic toxicity. The enzyme's activity is dependent on Ca²⁺ and Zn²⁺ and is inhibited by chelators such as EDTA. Its low levels of secondary proteases reduce the risk of non-specific protein degradation. The compound is intended for research use only.

[1]. Therapeutic applications of collagenase (metalloproteases): A review. Asian Pac J Trop Biomed, 2016, 6(11): 975-981.

Collagenase Type III is a research reagent used for the dissociation of specific tissues, particularly mammary glands, colon cancer stem cells, and breast epithelial tumor cells. Its low levels of secondary proteases make it suitable for applications where minimal damage to membrane proteins and receptors is desired.

812.418

9001-12-1

44384985

Yellow to brown solid

2

14

62

1490

12

CCCCCCCC[C@H]1CC[C@@]2([C@@]1(CC[C@]3(C2CC[C@@]4([C@@]3(CC5=C(C4)N=C6C[C@]7([C@@](CCC8[C@@]7(CC[C@]9([C@]8(CC[C@@H]9CCCCCCCC)C)C)C)(CC6=N5)C)C)C)C)C)C)C

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ≥ 100 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)