細胞アプリケーション

組織別 分散酵素適合表

組織別 分散酵素適合表

以下に示すアプリケーション情報はガイドラインであり、実験に使用するサンプルや測定エンドポイントにより、酵素タイプや濃度によって分散酵素の最適化を必要とする場合があります。

    SPECIES
    Human Rodent Porcine
CELL TYPE 繊維芽細胞 Liberase DH Research Grade
Protocol
Liberase DL Research Grade
Protocol
 
心筋細胞   Liberase DH Research Grade
Protocol
 
ケラチノサイト Liberase DH Research Grade
Protocol
Results
   
ランゲルハンス島   Liberase TL Research Grade
Protocol
Results
 
肝細胞   Liberase TM Research Grade
Protocol
Results
Liberase TM Research Grade
Protocol
腎臓   Liberase TM Research Grade
Protocol
Liberase DL Research Grade
Protocol
Results
結腸(上皮)癌 Liberase DH Research Grade
Liberase DL Research Grade
Protocol
Results
   
樹状細胞   Liberase TL Research Grade
Liberase DL Research Grade
Protocol
Results
 
幹細胞様初期前駆細胞   Liberase TL Research Grade
Protocol
Results
 
肺腫瘍 Liberase TM Research Grade
Protocol
Results
   
         



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注記:以下に紹介するアプリケーション データは多くがロシュ・ダイアグノスティックス社外で作成されたものであり、ロシュ・ダイアグノスティックス社の意見・見解を代表するものではありません。

Human Fibroblasts

Protocol

For more detailed information, refer to Isolation of Keratinocytes and Fibroblasts from Human Foreskin by One-Step Enzyme Incubation Using Liberase Research Grade Enzymes.

Human Keratinocytes

Protocol

For more detailed information, refer to Isolation of Keratinocytes and Fibroblasts from Human Foreskin by One-Step Enzyme Incubation Using Liberase Research Grade Enzymes.

Human Keratinocytes Viability and Functionality

Keratinocyte and fibroblast isolation experiments were performed in the laboratories of Dr. Andrea Heymer and Dr. Michaela Kaufmann at the Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Both cell types were isolated from human foreskin tissue, comparing the 2nd generation product Liberase DH Research Grade Enzyme with the formerly commercialized Liberase Blendzyme 1. The viability of the isolated cells was comparable for both enzymes (see Figure 1).

Cell viability of cells isolated with  Liberase DH Research Grade and Liberase Blendzyme 1

Figure 1: Cell viability of cells isolated with Liberase DH Research Grade and Liberase Blendzyme 1.

 

The functionality of the isolated keratinocytes was demonstrated by the development of a multilayered epidermis with a stratified closing layer, analogous to the stratum corneum.
Using cells isolated with Liberase Blendzyme 1, the epidermal layer consisted of 2-3 cell layers (see Figure 2A). In contrast, with the use of Liberase DH Research Grade Enzyme, the keratinocytes developed a 3-4 layered epidermis (see Figure 2C).
The early epidermal differentiation marker cytokeratin 10 was more expressed in skin equivalents fabricated with Liberase DH Research Grade Enzyme see (see Figure 2D) compared to Liberase Blendzyme 1 (BL1, see Figure 2B).

Functionality study of the isolated keratinocytes

Figure 2: Functionality study of the isolated keratinocytes.
(A) Layer formation of cells isolated with Liberase Blendzyme 1
(B) Expression of cytokeratin 10 in cells isolated with Liberase Blendzyme 1
(C) Layer formation of cells isolated with Liberase DH Research Grade Enzyme
(D) Expression of cytokeratin 10 in cells isolated with Liberase DH Research Grade Enzyme

*H&E = Hematoxylin and Eosin staining

Human Epithelial Colon Carcinoma Cells

Protocol

For more detailed information, refer to Isolation of Viable Epithelial Cells from Human Colon Carcinoma Tissue.

Human Epithelial Colon Carcinoma Cells Viability and Functionality

Liberase Research Grade enzymes were tested for the isolation of epithelial cells from colon carcinoma tissue samples. The goal was to identify the optimal enzyme blend for the specific tissue type and optimal conditions for dissociating this tissue type. Viable colon carcinoma cell yield was determined by double staining using a combination of two vital dyes, calcein-AM, as well as propidium iodide which discriminates viable and dead cells, and debris. The epithelial marker, BerEP4 was also used, showing that nearly 30% of the epithelial tumor cells survived the dissociation procedure using Liberase DH and DL Research Grade Enzyme Blends (see Figure 3).


Ratio of viable colon carcinoma epithelial cells expressed as a percentage of all epithelial cells after cell dissociation and isolation using Liberase DH and DL Research Grade Enzyme Blends

Figure 3: Ratio of viable colon carcinoma epithelial cells expressed as a percentage of all epithelial cells after cell dissociation and isolation using Liberase DH and DL Research Grade Enzyme Blends.

Data provided by Istvan Petak and Tamas Micsik, Semmelweis University, Budapest, Hungary.

Human Lung Tumor Cells

Protocol

Open access expiration: Detailed information is not available.

Human Lung Tumor Cells Viability

Liberase Research Grade enzymes were evaluated for the isolation of cells from human lung tumor tissue samples. The goal was to identify the optimal enzyme blend for the specific tissue type and optimal conditions for dissociating this tissue type. In two of three experiments, the proliferation capacity of isolated cells was higher in cultures from Liberase TM Research Grade compared to the competitor (see Figure 4).


Cell yield and viability after isolation of cells from human lung tumor tissue with Liberase TM Research Grade and a competitor's product

Figure 4: Cell yield (A) and viability (B) after isolation of cells from human lung tumor tissue with Liberase TM Research Grade and a competitor's product.

Data from Appelt et al., 2010, EuroBiotechNews Vol 9; Evaluation of Liberase TM Research Grade for cell isolation from human lung tumor tissue.

Rodent Fibroblasts

Protocol

Tissue biopsies (Sprague-Dawley rat skin tissue 1.0 cm x 1.0 cm) were chopped into small pieces with small surgical scissors and the addition of HEPES-Eagle's medium, containing Liberase DL Research Grade Enzyme at a final concentration of 500 µg/ml.
The prepared mixture was quickly placed in an atmosphere containing a mixture of oxygen and carbon dioxide (95% (v/v), Linde, Germany) and was incubated for 25 minutes at +37°C under agitation.

Next, the remaining tissue fragments were dissociated into even smaller pieces by up- and down-suction through different glass pipettes with progressively more restrictive orifices (20, 10. and 5 ml pipettes, respectively), and filtered through a nylon mesh (200 µm mesh). After centrifugation (800 x g for 5 minutes) and further purification by washing in Dulbecco's modified Eagle's medium (DMEM, Gibco, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria), 1 U/ml penicillin, and 10 mg/ml streptomycin (both PAA Laboratories, Austria), the cells were seeded into 25 cm2 flasks.

HEPES-Eagle's medium (pH 7.4): 10 mM HEPES buffer, 70% (v/v) modified Eagle's medium, 0.5% (v/v) Trasylol (Bayer AG, Germany), 1% (w/v) bovine serum albumin, 2.4 mM CaCl2.

Rodent Cardiac Myocytes

Protocol

Tissue biopsies (Sprague-Dawley rat heart tissue 1.0 cm x 1.0 cm) were chopped into small pieces with small surgical scissors and the addition of HEPES-Eagle's medium, containing Liberase DL Research Grade Enzyme at a final concentration of 200 µg/ml.
The prepared mixture was quickly placed in an atmosphere containing a mixture of oxygen and carbon dioxide (95% (v/v), Linde, Germany) and was incubated for 25 minutes at +37°C under agitation.

Next, the remaining tissue fragments were dissociated into even smaller pieces by up- and down-suction through different glass pipettes with progressively more restrictive orifices (20, 10. and 5 ml pipettes, respectively), and filtered through a nylon mesh (200 µm mesh). After centrifugation (800 x g for 5 minutes) and further purification by washing in Dulbecco's modified Eagle's medium (DMEM, Gibco, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria), 1 U/ml penicillin, and 10 mg/ml streptomycin (both PAA Laboratories, Austria), the cells were seeded into 25 cm2 flasks.

HEPES-Eagle's medium (pH 7.4): 10 mM HEPES buffer, 70% (v/v) modified Eagle's medium, 0.5% (v/v) Trasylol (Bayer AG, Germany), 1% (w/v) bovine serum albumin, 2.4 mM CaCl2.

Rodent Islet Cells

Protocol

Islets were isolated from adult male NMRI mice pancreata using Liberase TL Research Grade Enzyme as described in Nikolova G, Jabs N, Konstantinova I, et al., (2006); The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation, Dev Cell 10: 397 - 405.
Online reference (External Link)

Two milliliter of Liberase TL Research Grade Enzyme solution (1.081 WU/ml) was injected into the bile duct, followed by digestion at +37°C for different time intervals for optimization purposes. The enzymatic action was stopped using DMEM (Invitrogen GmbH, Karlsruhe, Germany) containing 15% FCS (PAA Laboratories GmbH, Coelbe, Germany). After subsequent washes, filtration (400 µm mesh), and gradient centrifugation (1100 x g for 30 minutes), islets were collected from the interphase between Histopaque 1077 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and DMEM. The collected islets were washed twice with DMEM and cultured overnight in uncoated petri dishes with CMRL (Invitrogen GmbH, Karlsruhe, Germany) media supplemented with 15% heat inactivated FCS, 100 U/ml penicillin with 0.1 mg/ml streptomycin (Invitrogen GmbH, Karlsruhe, Germany), 11 mM glucose, 0.15% NaHCO3 (Invitrogen GmbH, Karlsruhe, Germany), and incubated at +37°C and 5% CO2.

Rodent Islet Cells Viability and Functionality

In studies designed to investigate the influence of collagenase type on islet transplant success, rat islets were isolated with either Liberase RI Enzyme or Sigma Type V. When islets were transplanted into diabetic mice, fewer Liberase RI Enzyme-isolated islets were required to reverse diabetes, and they did so in a shorter period of time (see Figure 5).


Investigation of the influence of collagenase type on islet transplant success

Figure 5: Investigation of the influence of collagenase type on islet transplant success.

Data from Molano RD, Berney T, Ricordi C, Inverardi L. The effects of different enzyme formulations on the outcome of syngeneic transplants of marginal islet mass. Cell Transplantation 1999; 8(2):185.

 

Liberase Research Grade/digestion time

In a recent study comparing the 2nd generation Liberase Research Grade TL with Liberase RI Enzyme, a significant increase in islet yield in a shorter digestion time was demonstrated, while preserving the viability and functionality of the islets (see Figure 6).


Number of of  islets per mouse pancreas

Figure 6: Number of of islets per mouse pancreas.

Data from Yesil P. et al., 2009; A new collagenase blend increases the number of islets isolated from mouse pancreas, Islets 1:3, 185 - 190.
Online reference (External Link)

Rodent Hepatocytes

Protocol

1. Literature

Murine hepatocytes were isolated using 40 µg/ml Liberase TM Research grade following the protocols described by P.O. Seglen, Preparation of isolated rat liver cells. In: D.M. Prescott Editor, Methods in Cell Biology Academic Press, New York (1976), pp. 29–83.
Online reference (External Link)

 

2. Stock Solutions

Glucose solution KH buffer HEPES buffer
D-glucose 9 g/l NaCl
KCl
KH2PO4
pH 7.6 with NaOH

60 g/l

1.75 g/l

1.6 g/l

HEPES 60 g/l; pH 8.5 with NaOH
     
Glutamine solution Insulin solution EGTA solution
L-glutamine 7 g/l, freshly prepared Insulin 2 g/l dissolved in 1 N NaOH, adjusted to pH 7.6 by 1 N HCl; freshly prepared EGTA 47.5 g/l dissolved by addition of NaOH, adjusted to pH 7.6 by HCl
     
CaCl2 solution MgSO4 solution  
CaCl2 x 2 H2O 19 g/l MgSO4 x 7 H2O 24.6 g/l  
     
Amino acid solution
L-alanine 0.27 g/l L-lysine 1.30 g/l
L-aspartic acid 0.14 g/l L-methionine 0.55 g/l
L-asparagine 0.4 g/l L-ornithine 0.65 g/l
L-citrulline 0.27 g/l L-phenylalanine 0.55 g/l
L-cysteine 0.14 g/l L-proline 0.55 g/l
L-histidine 1.0 g/l L-serine 0.65 g/l
L-glutamic acid 1.0 g/l L-threonine 1.35 g/l
L-glycin 1.0 g/l L-tryptophan 0.65 g/l
L-isoleucine 0.40 g/l L-tyrosine 0.55 g/l
L-leucine 0.8 g/l    
       

Dissolve amino acids that can not be dissolved at neutral pH by addition of 10 N NaOH, and thereafter adjust to pH 7.6 by 37% HCl.

 

3. Working Solutions, each for 1 mouse

EGTA buffer Collagenase buffer Suspension buffer
  • 62 ml glucose solution
  • 10 ml KH buffer
  • 10 ml HEPES buffer
  • 15 ml amino acid solution
  • 1 ml glutamine solution
  • 0.4 ml EGTA solution
  • 38.75 ml glucose solution
  • 6.25 ml KH buffer
  • 6.25 ml HEPES buffer
  • 9.5 ml amino acid solution
  • 2.5 ml CaCl2 solution
  • 0.625 ml glutamine solution
  • 40 µg/ml Liberase TM Research Grade

dissolve Liberase Enzyme in the prewarmed mixture of the above mentioned solutions immediately before use.

  • 68.85 ml glucose solution
  • 11.1 ml KH buffer
  • 11.1 ml HEPES buffer
  • 16.65 ml amino acid solution
  • 1.1 ml glutamine solution
  • 0.865 ml CaCl2 solution
  • 0.435 ml MgSO4 solution
  • 0.222 g BSA

dissolve BSA in the mixture of the above mentioned solutions immediately before use.

     

 

4. Murine Liver Cell Isolation Procedure

8-12 weeks old NMRI mice were anesthetized with an i.p. injection of 150 mg/kg pentobarbital. A circulation with warm water kept the buffer flow at a constant temperature of +37°C. All used buffers that came into contact with hepatocytes were prewarmed to +37°C.

The liver was perfused in situ via the inferior vena cava for at most 10 minutes with EGTA buffer while vena porta was used as outflow. During the total isolation, the flow rate was 6 ml/minute and the pressure of perfusion did not exceed a 20 cm water column. The perfusion was continued with collagenase buffer for not longer than 10 minutes.

After digestion, the liver was removed from the animal, the gallbladder was abscised, and then the liver was dissociated carefully in cold suspension buffer. Afterwards, the liver cell suspension was filtered through gauze (100 µm) and centrifuged for 5 minutes at 31 x g and +4°C. Two more washing steps with suspension buffer were done; the hepatocytes were centrifuged again and were then resuspended in a defined volume of medium. Using a 1:10 dilution with trypan blue, the vitality of the hepatocyte suspension was determined.

Rodent Hepatocytes Viability

Hepatocyte isolation experiments were performed in the laboratory of Isabell Schulz (University of Leipzig, Germany). Liberase TM Research Grade was used in a side-by-side comparison with a competitor's collagenase, routinely used in the laboratories established and optimized working protocol. The excellent performance of Liberase TM Research Grade for mouse hepatocyte isolation is demonstrated by analysis of vitality and functionality.

For more detailed information, refer to Increased Vitality of Primary Mouse Hepatocytes Isolated with Liberase TM Research Grade Enzymes.

 

Cell Yield and Vitality

Cell yield (see Figure 7) and vitality (see Figure 8) of primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase. Eight single preparations were performed.

Cell yield of primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase.

Figure 7: Cell yield of primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase.

Cell vitality of primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase

Figure 8: Cell vitality of primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase.

 

Functional Analysis

Functional analysis of primary mouse hepatocytes isolated with Liberase TM Research Grade and a competitor’s collagenase. The amount of the metabolic marker urea secreted into the supernatant was measured after 1, 3, 5, and 7 days in cell culture (see Figure 9). Analysis of the amount of albumin after 1, 3, and 5 days of culture (see Figure 10). Two separate cell preparations were investigated.

Amount of urea secreted by primary mouse hepatocytes after isolation with Liberase TM Research  Grade and a competitor's collagenase

Figure 9: Amount of urea secreted by primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase.

Amount of albumin secreted by primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase

Figure 10: Amount of albumin secreted by primary mouse hepatocytes after isolation with Liberase TM Research Grade and a competitor’s collagenase.

Rodent Kidney Cells

Protocol

Tissue biopsies (Sprague-Dawley rat kidney tissue 1.0 cm x 1.0 cm) were chopped into small pieces with small surgical scissors and the addition of HEPES-Eagle's medium, containing Liberase TM Research Grade Enzyme at a final concentration of 100 µg/ml.
The prepared mixture was quickly placed in an atmosphere containing a mixture of oxygen and carbon dioxide (95% (v/v), Linde, Germany) and was incubated for 25 minutes at +37°C under agitation.

Next, the remaining tissue fragments were dissociated into even smaller pieces by up- and down-suction through different glass pipettes with progressively more restrictive orifices (20, 10. and 5 ml pipettes, respectively), and filtered through a nylon mesh (200 µm mesh). After centrifugation (800 x g for 5 minutes) and further purification by washing in Dulbecco's modified Eagle's medium (DMEM, Gibco, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria), 1 U/ml penicillin, and 10 mg/ml streptomycin (both PAA Laboratories, Austria), the cells were seeded into 25 cm2 flasks.

HEPES-Eagle's medium (pH 7.4): 10 mM HEPES buffer, 70% (v/v) modified Eagle's medium, 0.5% (v/v) Trasylol (Bayer AG, Germany), 1% (w/v) bovine serum albumin, 2.4 mM CaCl2.

Rodent Dendritic Cells

Protocol

Preparation of Liberase Enzyme-digested spleenocyte suspension for dendritic cell isolation from C57BL/6 mice.

  • Prepare Liberase Enzyme/DNase I solution (total volume 1 - 2 ml/spleen): Liberase (1.67 Wünsch units/ml) + DNase I (0.2 mg/ml; Roche Diagnostics) in LPS-free RPMI. Both Liberases DL and TL Research Grade gave good results.
  • Inject 1 - 2 ml of this solution while holding the spleen with a pair of curved forceps above a 15 ml Falcon tube (25G needle; choose 3 - 5 sites along the spleen). Some of the solution will be retained in the spleen (note the swelling) but most of it will drain through, releasing many cells (note color change).
  • Let spleen slide to the bottom of the tube, completely immersing it into the Liberase Enzyme/DNase I solution. Incubate 20 - 30 minutes at +37°C.
  • Filter cell suspension and digested spleen through a strainer into a fresh 50 ml Falcon tube. Wash the spleen inside the strainer once with PBS/0.5 mM EDTA. If necessary, release cells from the spleen by gently grinding it with a plunger (1 ml syringe), followed by several washings with PBS/0.5 mM EDTA. Repeat grinding and washing steps until most of the spleen tissue has been eluted into the tube.
  • Add PBS/0.5 mM EDTA to a final volume of 50 ml, remove an aliquot for cell counting, and spin at +4°C (1400 x g, 5 minutes). Expect ≥ 10e8 cells/spleen.
  • Either fix cells or resuspend in buffer of choice:
    a) RPMI + 10% FCS for cell culture
    b) FACS buffer for FACS staining
    c) MACS buffer for isolation of CD11c + DC

Rodent Dendritic Cells Results

Spleens from C57BL/6 mice were digested with different Liberase enzyme blends for the isolation of dendritic cells. A Liberase Enzyme/DNase I solution (total volume 1 - 2 ml/spleen): Liberase (1.67 Wünsch units/ml) + DNase I (0.2 mg/ml; Roche Diagnostics) in LPS-free RPMI was used.

The percentage of CD8-positive dendritic cells (CD11c+ MHCII+) was compared by flow cytometry after digestion with Liberase CI, Liberase DL Research Grade, and Liberase TL Research Grade enzymes (see Figure 11). Both, Liberase TL and DL Research Grade enzymes gave optimal results. The results are representative for three spleen digestions performed in two independent experiments.


Flow cytometry plots of Roche Liberase enzymes

Figure 11: Flow cytometry plots. Averages and standard deviations are indicated. Gating is shown (FCS forward scatter).

Data from Brocker laboratory, Institute for Immunology, Ludwig Maximilians University, Munich.

Rodent Stem-Cell Like Early Progenitors

Protocol

For more detailed information, refer to Successful Isolation of a Proliferating Cell Population with Stem Cell-Like Properties from Rat Pancreas Using Liberase TL Research Grade.

Rodent Stem-Cell Like Early Progenitors Results

The performance of Liberase TL Research Grade for the isolation of early progenitor cells from rat pancreatic tissue was analyzed. Tissue digestion with Liberase TL Research Grade resulted in high yields of isolated cells. In addition, markers for the three germ layers ecto-, endo-, and mesoderm, as well as for early stem cells, were detected. Impedance curves of the isolated progenitor cells were recorded in real time using the xCELLigence System.


Determining the optimal Liberase TL Research Grade concentration

Figure 12: Determining the optimal Liberase TL Research Grade concentration. Comparison of the isolated number of viable cells (a) and the number of viable, proliferating adherent cells after 5 days of culture (b). Please note that the demonstrated results were derived from only one experiment and had to be statistically corrobated.

Immunocytochemical analysis of isolated cells

Figure 13: Immunocytochemical analysis of isolated cells.
Left: Proteins characteristic for stem cells (Nanog, Nucleostemin), endodermal cells (GATA4), mesodermal cells (α-SMA), and ectodermal cells (GFAP) were detected.
Right: As a control, the cell nuclei were stained with DAPI.

Porcine Hepatocytes

Protocol

Tissue biopsies (porcine liver tissue 1.5 cm x 1.5 cm) were chopped into small pieces with small surgical scissors, and the addition of HEPES-Eagle's medium, containing Liberase TM Research Grade Enzyme at a final concentration of 25 µg/ml. The prepared mixture was quickly placed in an atmosphere containing a mixture of oxygen and carbon dioxide (95% (v/v), Linde, Germany) and was incubated for 25 minutes at +37°C under agitation.

Next, the remaining tissue fragments were dissociated into even smaller pieces by up- and down-suction through different glass pipettes with progressively more restrictive orifices (20, 10, and 5 ml pipettes, respectively), and filtered through a nylon mesh (200 µm mesh). After centrifugation, (800 x g for 5 minutes) and further purification by washing in Dulbecco's modified Eagle's medium (DMEM, Gibco, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria), 1 U/ml penicillin, and 10 mg/ml streptomycin (both PAA Laboratories, Austria), the cells were seeded into 25 cm2 flasks.

HEPES-Eagle's medium (pH 7.4): 10 mM HEPES buffer, 70% (v/v) modified Eagle's medium, 0.5% (v/v) Trasylol (Bayer AG, Germany), 1% (w/v) bovine serum albumin, 2.4 mM CaCl2.

Porcine Kidney Cells

Protocol

Tissue biopsies (pig kidney tissue 1.5 cm x 1.5 cm) were chopped into small pieces with small surgical scissors, and the addition of HEPES-Eagle's medium, containing Liberase TM Research Grade Enzyme at a final concentration of 100 µg/ml. The prepared mixture was quickly placed in an atmosphere containing a mixture of oxygen and carbon dioxide (95% (v/v), Linde, Germany) and was incubated for 25 minutes at +37°C under agitation.

Next, the remaining tissue fragments were dissociated into even smaller pieces by up- and down-suction through different glass pipettes with progressively more restrictive orifices (20, 10, and 5 ml pipettes, respectively), and filtered through a nylon mesh (200 µm mesh). After centrifugation, (800 x g for 5 minutes) and further purification by washing in Dulbecco's modified Eagle's medium (DMEM, Gibco, Germany) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria), 1 U/ml penicillin, and 10 mg/ml streptomycin (both PAA Laboratories, Austria), the cells were seeded into 25 cm2 flasks.

HEPES-Eagle's medium (pH 7.4): 10 mM HEPES buffer, 70% (v/v) modified Eagle's medium, 0.5% (v/v) Trasylol (Bayer AG, Germany), 1% (w/v) bovine serum albumin, 2.4 mM CaCl2.

Porcine Kidney Cells Viability

Cell isolation experiments from porcine kidney were done in the laboratory of Professor Charli Kruse (Fraunhofer EMB Lübeck, Germany).
The experimental objective was to come to a side-by-side performance comparison of the new Liberase Research Grade Enzyme Blends to the previous generation Liberase Enzymes.
Figure 14 shows the viability of porcine nephrocytes isolated using the new generation Liberase DL Research Grade, Liberase TM Research Grade, Liberase TH Research Grade, and the old Liberase Blendzyme 3 enzymes. The cell yield of two separate isolations was determined by automatic counting after 2 days of culture.


Viability of porcine nephrocytes isolated using the new generation Liberase DL Research Grade, Liberase TM Research Grade, Liberase TH Research Grade, and the old Liberase Blendzyme 3 enzymes

Figure 14: Viability of porcine nephrocytes isolated using the new generation Liberase DL Research Grade, Liberase TM Research Grade, Liberase TH Research Grade, and the old Liberase Blendzyme 3 enzymes.

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