Liberase™ TM Research Grade Protocol & Troubleshooting

Product No. LIBTM-RO

Protocol

Selection of the appropriate Liberase Research Grade Enzyme Blend

Your current tissue dissociation protocol, and the extent to which you have been successful with traditional collagenase are good guides for selecting the appropriate Liberase Enzyme Blend. Select the Liberase Enzyme Blend most similar to your tissue dissociation application, or based upon a specific manufacturer and collagenase type. Please refer to the “Liberase Research Grade Enzyme Blends Technical Tip - April 2010” , or go to the Roche Special Interest Site Tissue Dissociation. The continually updated table contains a wide variety of tissue dissociation applications, and the recommended Liberase Enzyme Blends, and their starting concentrations. The table also provides links to either full text protocols or citations for each application.

Liberase Research Grade: Protocols for isolation of various cell types

For protocols using the Liberase Research Grade Enzyme Blends, please visit the Roche Special Interest Site Tissue Dissociation.The following working procedures have been routinely and successfully used in the Roche R&D departments for the isolation of:

• Hepatocytes from rat liver
• Pancreatic islets from rat and mouse pancreas
• Adipocytes from rat epididymal fat pads
• Endothelial cells from human umbilical vein
• Endothelial cells from porcine and bovine aorta
• Cardiomyocytes from rat heart

Please note that the working concentrations for the particular enzymes given in the following above procedures general guidelines that should be adapted to individual requirements.At the end of each protocol, references provide access to the relevant literature.

Isolation of Rat Hepatocytes

For the isolation of rat hepatocytes collagenase H is recommended. To obtain a high yield of intact cells (up to 95%), perfusion of the liver with collagenase is the method of choice^1-3. The perfusion is carried out in two steps through the hepatic portal vein and involves initially (first step) a calcium-removing solution (Ca²⁺-free and/ or containing EGTA) followed by a collagenase-containing medium (second step). Since collagenase requires Ca²⁺, it should be present during the second step.

Equipment and perfusion apparatus

Detailed information concerning equipment and the perfusion apparatus is given in Reference 2.

Solutions

Perfusion medium (20 x; stock solution): NaCI, 138.0 g/l; KCI, 8.9 g/l; Na₂HP04, 11.4 g/l; NaH₂P04, 5.4 g/l; glucose, 180.0 g/l; MgS0₄ x 7 H₂0, 6.0 g/l; sterile. Perfusion medium (1 x working solution; 1 I per liver): 50 ml perfusion medium (20 x); 22.5 ml sterile NaHCO₃, 7.5%; 2 ml sterile phenol red, 0.5%; fill up to 1000 ml with sterile redistilled water; adjust to pH 7.4 (with 1 N HCI or 1 N NaOH). This medium is recommended for perfusion of the liver and washing the cells. For the first perfusion step, EGTA, 0.08 g/l (0.2 mmol/l, sterile), may be added to the perfusion medium (1 x) (see Reference 2, 4).For the second perfusion step prepare Collagenase H solution to be added to the perfusion medium (1 x). To do this, collagenase H, 100 mg, is dissolved in 4 ml perfusion medium (1 x) (final concentration 25 mg/mL) and filtered (0.2 μm-filter). To activate the collagenase, 2.5 ml sterile CaCI₂ solution, 200 mmol/l, should be added to 100 ml collagenase perfusion medium.

Culture medium

DMEM (1.1 g/l glucose) containing MEM vitamins 1 x; MEM amino acids 1 x; non-essential amino acids 1 x; glutamine, 2 mmol/l; lactate (pH 7.4), 1 %; penicillin/ streptomycin, 1 %. Add fetal calf serum (FCS), 5%, immediately before plating the cells. For subculture add dexamethasone, 10^-4 mmol/l and insulin, 2 x 10^-5 mmol/l to the culture medium. For serum-free subculture, FCS should be replaced by BSA, 1 mg/mL. These medium formulations can be used after the first medium exchange (90 min. after plating).

Collagen-coating solution: Dissolve 20 mg collagen, e.g., collagen (type I) or collagen (rat tail), in 10 ml sterile acetic acid, 0.1 N (final concentration 2 mg/mL) or use sterile collagen solution, e.g. collagen S., Sodium pentobarbital, e.g. Nembutal^®, Sodium heparin, e.g., Liquemin^® 25000^®

Procedure

Anesthetize male Spraque-Dawley rats (body weight approx. 200 g) with 50 μg Nembutal^® /g living weight (i.p.) and open the abdomen. Spray liver and intestine with approx. 0.5 ml liquemin to avoid coagulation. Uncover the liver and inject a cannula into the portal vein. Start perfusion (first step) with perfusion medium Ca²⁺-free, with a pressure of 50 ml of H₂O. Discard the first 50 ml to remove erythrocytes and plasma proteins. Remove the liver and transfer it to a perfusion apparatus, which maintains the perfusate temperature at +37 °C whilst constantly oxygenating it. Perfuse the liver (recirculating via backflow through the caval vein) with 100 ml perfusion medium (1x) oxygenated with carbogene (95% O₂, 5% CO₂) at a flow rate of 40 ml/min for 5 min. Add sterile collagenase solution (4 ml of a 25 mg/mL solution) to the perfusion medium (final concentration approx. 1 mg/mL) and continue the perfusion for 10 to 30 minutes (second step). Perfusion is terminated when the liver shows a soft consistency (check by finger pressure).

Subsequent to perfusion, place the liver into a sterile 50 mm petri dish containing about 20 ml perfusion medium (1 x), disrupt the liver capsula with two sterile forceps and dissociate liver cells by gently shaking the organ. Remove non-dissociated tissue pieces and filter the cell suspension through a 74 μm filter or 4 sheets sterile gauze respectively. Collect the filtrate in a sterile beaker and fill up to 200 ml with perfusion medium (1 x; oxygenated with carbogen). Spin the cell suspension in 50 ml portions for 60 seconds (50 x g, room temperature) and resuspend the cells in perfusion medium (1 x; oxygenated with carbogen) reducing the volume by 50%. Repeat this washing procedure twice, each time reducing the volume by 50%. Resuspend the cells in culture medium containing FCS and seed 3 x 10⁵ cells/ml in collagen-coated (2.5 μL/cm² of a 2 mg/mL collagen-coating solution) culture vessels. Incubate at +37 °C with 5% CO₂. Remove culture medium and non-attached cells 90 min. after plating. For further cell culture (subculture), serum free culture medium can be used.

Liberase Research Grade: Skin tissue

External Roche partners from the Fraunhofer Institute in Stuttgart, Germany have systematically examined Liberease Research Grade Enzyme Blends for the isolation of keratinocytes and fibroblasts from the epidermis of human foreskin tissue. Please visit the Roche Special Interest Site Tissue Dissociation for further information.

Liberase Research Grade recommended for the rat cardiac myocytes

Approximately 200 μg/ml Liberase DH Research Grade is recommended. Please find a protocol on our Special Interest Site Tissue Dissociation. In addition, many other protocols are provided in the Liberase database.

Liberase Research Grade: Dissociation of fatty tissue

Application data for the isolation of cells from fatty tissue using the second generation Liberase Research Grade products are not available. There is however a protocol for the isolation of adipocytes using the first generation product, Liberase Blendzyme 3, at the Roche Special Interest Site Tissue Dissociation.

The section "Transition from previous Liberase Enzyme Blends" may help to select the corresponding new product that easily converts the concentration.

Liberase Research Grade: Paraffin embedded tissue

The isolation of cells from paraffin embedded tissue using Liberase Enzymes has not been evaluated.

Detection of extracellular antigens

A protocol for dendritic cells from spleen is available on the Roche Special Interest Site Tissue Dissociation. To develop this protocol all five new Liberase Research Grade Enzyme Blends were tested . The best results were obtained using Liberase TL and DL Research Grade Enzyme Blends. Antigens, such as MHCII, DC11c and CD8, could be detected.

Note: All the above protocols were developed by customers and have not been validated by Roche. Roche can not be made liable for the contents and the successful execution of the protocols. Roche provides customer protocols on the Roche homepage as guidelines and a starting point, and as a service facilitating the exchange of scientific information within the research community.

Cell dissociation from rodent neonatal brain tissue just prior to FACS sorting

Roche does not offer a protocol for isolating neonatal rodent brain tissue using Liberase Research Grade Enzyme Blends. As a starting point for this application, the Liberase DH and DL Research Grade would be the most gentle products, since the neutral protease provided in these Liberase Enzyme Blends is Dispase^®. Please refer to the Roche Special Interest Site for more detailed product information.

Cell isolation from lung tumor tissue using Liberase TM Research Grade

Refer to the publication on this Roche Applied Science Special Interest Site, http://www.roche-applied-science.com/sis/collagenase/col_docs/EBN2010_09_Roche.pdf for experimental details for isolating cells from lung tumor tissue. As a starting point, a 10 ml working solution with 0.25 Wünsch Units/ml Liberase TM Research Grade was used per gram lung tissue.

Troubleshooting

Enzyme Composition

All Liberase Research Grade Enzyme Blends consist of a mixture of collagenase I and II and a neutral protease. Liberase TL, TM and TH Research Grade contain collagenase I, II and the neutral protease thermolysin. Liberase DL and DH Research Grade are composed of collagenase I, II and the neutral protease Dispase^®.

Visit the Roche Special Interest Site Tissue Dissociation to learn how these enzyme blends differ in their aggressiveness and to identify the appropriate Liberase Research Grade for your application. If your application is not included in our current database, please go to the next section, "Liberase TM Research Grade." You can also select a Liberase Research Grade Enzyme Blend based upon your experience with the 1^st generation Liberase Blendzymes and with traditional collagenases using the Liberase enzyme selection guides.

Collagenase and Liberase Blendzymes: The differences

Traditional collagenase preparations are isolated from bacterial (Clostridium histolyticum) culture supernatants. These preparations are heterogeneous, containing as many as 30 different enzymes, cellular debris, pigments and endotoxin. Endotoxin levels and variability are the most significant liabilities of traditional collagenase. Liberase enzymes offer many advantages that distinguish them significantly from traditional collagenases. Because each Liberase purified enzyme blend has been specifically formulated per application using response surface modeling, optimal results can be expected in terms of:

• Increased cell yield
• Improved cell viability
• Greater cell functionality

Each Liberase lot has the same enzyme activity specifications. Each lot is tested for endotoxin to ensure consistently low levels. There is no longer a need to perform lot selection or whole batch reservation. Each lot is guaranteed to perform the same in any given tissue dissociation protocol.

Please refer to the Special Interest Site Tissue Dissociation for more detailed information.

RNAse contamination

RNase-free is not a quality control criterion of the Liberase production procedure. During tissue dissociation, cells are disrupted and RNases are released. For this reason, RNase-free quality does not provide additional benefit.

Liberase Research Grade components are highly purified preparations. HPLC chromatograms show there are no other proteins other than Collagenase I and II and the neutral Protease.

Liberase Research Grade Certificates of Analysis

The Certificates of Analysis (CoAs) for Liberase Research Grade products, 2nd generation, indicate the amounts of Col Ia, Col I, and Col II, endotoxin levels and lyophilizate appearance.

First generation Liberase blends were released using a more variable activity assay. The activity values were provided in the CoA. In contrast, 2nd generation Liberase Research Grade products, are released based on protein amounts (HPLC and protein mass) and have higher lot-to-lot consistency.

The activity test for the neutral protease has also changed in 2nd generation Liberase products. FITC casein units are no longer measured. The FITC test has been replaced by a non-fluorescent activity assay. Amounts of neutral protease have not changed. The activities of neutral protease determined using the new test, correspond to the FITC units determined previously.

Collagenase/Liberase Research Grade Enzyme Blends: Categorization into different types

Bacterial collagenase, or more accurately clostridiopeptidase A, is a protease with a specificity for the X-Gly bond in the sequence Pro-X-Gly-Pro, where X is most frequently a neutral amino acid. Such sequences are found in high frequency in collagen, and only rarely in other proteins. Purified clostridiopeptidase A is ineffective in dissociating tissues due to incomplete hydrolysis of collagen polypeptides and its limited activity against high concentrations of non-collagen proteins and other macro-molecules found in the extracellular matrix. The collagenase most commonly used for tissue dissociation is a crude preparation from the bacteria Clostridium histolyticum containing clostridiopeptidase A, in addition to a number of other proteases and lipases.

Crude collagenases are a mixture of different enzymatic activities making them the basis for several commercially available collagenases. These collagenase types vary in their relative amount of collagenase and other proteases, including clostripain, one with a trypsin-like activity and a neutral protease. For example, Roche′s Collagenase A, B and D contain different ratios of the various proteolytic activities, while Collagenase H and P are function-tested for the isolation of hepatocytes (H) and pancreatic islets (P) from rat. This facilitates selection of the preparation best suited for the dissociation of a particular tissue.

Roche Collagenase is assayed in Wünsch units (1 pmol of product formed per minute at +25 °C with Wünsch substrate). Frequently, collagenase activities are given in Mandl units (1 μmol leucine liberated from collagen in 5 h at +37 °C). To date, there is no accepted conversion factor for these two units of activity, since the unit depends, in part, on the concentration of contaminating proteases in the collagenase preparation, an indefinable variable. The differences in unit definition and composition of enzyme activities make it complicated to exactly compare or convert collagenases from different suppliers.Please refer to the “Liberase Research Grade Enzyme Blends Technical Tip - April 2010” for more detailed information.

Reconstitution:

1. Increase the volume of the reconstitution buffer by 2-fold.
2. Vortex shortly.
   

Optimize Your Tissue Dissociation Procedure:

Important points for optimization:

• Liberase Research Grade Purified Enzyme Blends contain only collagenase and neutral protease.
• Collagenase enzymes digest the intercellular matrix.
• Neutral proteases act synergistically with collagenase.
• Given sufficient time and concentration, neutral proteases damage cell surface proteins.
• Time of dissociation, enzyme ratios, and enzyme concentration all affect the tissue-dissociation outcome.
• Use Liberase Research Grade Purified Enzyme Blends without modifying factors, such as serum, BSA, or protease inhibitors.

Note whether the yield, viability, or functionality of your cells isolated with Liberase enzyme is less than optimal. Find the probable cause, then act on the recommendation. Refer to enzyme mixture agressiveness described in “Biological Activity” for information on neutral protease specific activity increasing within the Liberase Research Grade panel.

Observation 1	Observation 2	Possible cause	Recommendation
Low cell viability	Dissociation very rapid	Enzyme concentration too high	Reduce enzyme concentration 50%.
		Enzyme mixture aggressiveness too high	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
	Dissociation very slow	Enzyme concentration too low	Increase enzyme concentration 50%.
		Enzyme mixture aggressiveness too low	Select a Liberase Research Grade Purified Enzyme Blend containing higher amounts of neutral protease.
Impaired cell function	Cell viability >80%, cell yield is reasonable	Reduce enzyme concentration 25%.
		Enzyme mixture aggressiveness too high	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
Low cell yield	Cell viability >80%	Enzyme concentration too low	Increase enzyme concentration 25-50%.
		Enzyme mixture aggressiveness too low	Select a Liberase Research Grade Purified Enzyme Blend containing higher amounts of neutral protease.
	Cell viability <80%	Enzyme concentration too high	Reduce enzyme concentration 50%.
		Enzyme mixture aggressiveness too high	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
		Mechanical (shear) force is excessive	Reduce shear force in all aspects of dissociation. Treat tissue gently.
Released cells clump in gelatinous stringy form	Cell yield and viability are acceptable	DNA release, subsequent to cell lysis, is causing clumping	More prevalent in some tissues. If cell viability is acceptable, add DNase to dissociation mixture.
	Cell yield or viability are reduced	Mechanical (shear) force is excessive	Reduce shear force in all aspects of dissociation. Treat tissue gently.

References

1. Berry NM and Friend SD, 1969, Isolated rat hepatocytes, J. Cell. Biol., 43, 506 - 520.

2. Seglen, P.O, 1976, Preparation of isolated rxat liver cells, Methods Cell Biol., 13, 29-83.

3. Schlepper-Schäfer J, Kolb-Bachofen V, Kolb H. 1980. Analysis of lectin-dependent recognition of desialylated erythrocytes by Kupffer cells. 186(3):827-831. https://doi.org/10.1042/bj1860827

4. Laishes BA, Williams GM. 1976. Conditions affecting primary cell cultures of functional adult rat hepatocytes. In Vitro Cell.Dev.Biol.-Plant. 12(7):521-532. https://doi.org/10.1007/bf02796495

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