Application Note | ELASTOSENS™ BIO
How to measure the enzymatic degradation of tissues and organs using the ElastoSens™ Bio?
by Dimitria Camasão and Assala Larbes,
Application Scientists, Rheolution Inc.
In colaboration with
Nordmark Biochemicals
SUMMARY
- ElastoSens™ Bio delivers precise measurements on the enzymatic degradation of native tissue that can serve to optimize the yield of cell extraction.
- Enzymes are commonly used in the field of regenerative medicine to degrade native tissues for the extraction of cells and other components, or as a part of physiological-like fluids to mimic in vivo conditions for biomaterials in development.
- The performance of enzymes can be measured in terms of weight loss or mechanical properties.
- The viscoelastic properties of kidney tissue during incubation with collagenase was precisely measured using the ElastoSens™ Bio.
- The increasing concentration of enzymes in solution accelerated the degradation of kidney samples.
INTRODUCTION
Enzymes such as collagenase are used to extract cells and other components from native tissues due to their ability to selectively break down extracellular matrix components. The released cell populations are used in various research and therapeutic applications, such as cell culture, cell therapy, and regenerative medicine. Cell extraction yield for a given tissue type and source depends on the type of enzyme and other components selected for the degradation process, their dosage, the time exposure to the degrading solution (to avoid harming cells), among others.
Additionally, enzymes are also used to compose physiological-like fluids that simulate in vivo conditions. These biomimetic environments enable researchers to test and refine their biomaterials under conditions closely resembling the human body, ultimately leading to more effective and clinically relevant regenerative therapies.
The measurement of the substrate’s weight loss and mechanical properties are two common methods to assess the performance of enzymes and to characterize degradation. Table 1 summarizes the differences in some practical aspects between the weight loss technique and the mechanical testing using the ElastoSens™ Bio. Overall, weight loss can lack consistency and sensitivity which can be provided with the mechanical testing.
Table 1: Practical differences between methodologies for the characterization of tissues and organs degradation (weight loss and mechanical measurements with the ElastoSens™ Bio) during the exposure to enzymes.
In this study, the degradation of kidney tissue due to the exposure to collagenase was evaluated through its viscoelastic properties non-destructively measured (during the whole experiment) using the ElastoSens™ Bio. Different concentrations of the enzyme were tested to analyze the consistency and the sensitivity of this method to characterize the degradation of tissues.
MATERIALS AND METHODS
Calf kidney was minced, and 0.3 g was added into the µ-volume sample holder of the ElastoSens™ Bio following the methodology 2 previously described (Figure 1A-B). Collagenase NB4G (Nordmark, Germany) was dissolved in PBS at the concentration of 0.15 U/mL, 0.5 U/mL and 1.5 U/mL. Prior to the first test on the ElastoSens™ Bio (test at t0), the prepared samples were immersed in pure PBS at 37 °C for 20 min. Then, the samples were incubated into 3 mL of the collagenase solutions (n=5 for each collagenase concentration) in a 12-well plate at 37 °C for a total of 1 hour (Figure 1C). Every 15 min, the samples were taken and tested in the ElastoSensTM Bio for 2 min at 37 °C (and re-incubated again after testing). Average results are expressed as mean ± standard deviation. Comparisons among groups were evaluated by one-way ANOVA with post-hoc Tukey test to correct for multiple comparisons. Significance was retained when p < 0.05.
Figure 1: Minced kidney (0.3 g) introduced into the µ-volume sample holder following the methodology 2 (A-B), and sample holders incubated with the collagenase solution in the 12-well plate (C).
RESULTS AND DISCUSSION
Figure 2 illustrates the effect of the collagenase NB4G (0.5 U/mL) on the shear storage modulus (G’) of the kidney samples at each 15 min during the 1-hour incubation. The G’ of the samples gradually decreased during the exposure to collagenase reaching values lower than 40% of its original G’. It is interesting to note the good repeatability of the results especially considering the internal heterogeneity of organs (standard deviations of approximately 10%).
Figure 2: Shear storage modulus (G’) of kidney samples as a function of exposure time to collagenase NB4G (0.5 U/mL) in absolute and relative values.
Figure 3 displays the decrease of G’ in relation to t0 (G’ of the kidney samples before exposure to collagenase) for the three concentrations of collagenase tested. It was found that the increase in enzyme concentration accelerated tissue degradation, showing statistical differences already after 15 min of incubation (p<0.05). Kidney tissue reached 51.8 ± 9.6 %, 39.1 ± 3.4 %, and 24.5 ± 3.4 % of its original G’ after 1-hour incubation with collagenase NB4G at 0.15 U/mL, 0.5 U/mL and 1.5 U/mL, respectively.
Figure 3: Relative shear storage modulus (G’, %) of kidney samples as a function of exposure time to collagenase NB4G at 0.15 U/mL (blue), 0.5 U/mL (turquoise) and 1.5 U/mL (orange).
CONCLUSIONS
Native tissue cut into small pieces were successfully loaded and tested into the µ-volume sample holder of the ElastoSens™ Bio. The use of this holder facilitates an efficient enzyme action by maximizing the sample’s surface area exposed to the enzymatic solution within a 12-well plate. The exposure of kidney samples to collagenase NB4G led to a gradual degradation precisely measured by the ElastoSens™ Bio in terms of viscoelastic properties.
Impact & Use
- Developing an easy and consistent protocol for precisely measuring the degradation of tissues and biomaterials caused by exposure to degrading solutions.
- Comparing the performance of different enzymes and extraction buffers on the target tissues or biomaterials.
- Optimizing the dosage of enzymes and other components to achieve desired degradation time and extent, enhancing cell extraction yields.
- Identifying cost-effective enzyme candidates, dosages, and durations to reduce the overall degradation process cost.
- Ensuring the batch-to-batch consistency and efficacy of an enzyme.
- Optimizing the performance of enzymes and extraction buffers to specific tissues and requirements.
ElastoSens™ Bio
INTRODUCTION
IEnzymes such as collagenase are used to extract cells and other components from native tissues due to their ability to selectively break down extracellular matrix components. The released cell populations are used in various research and therapeutic applications, such as cell culture, cell therapy, and regenerative medicine. Cell extraction yield for a given tissue type and source depends on the type of enzyme and other components selected for the degradation process, their dosage, the time exposure to the degrading solution (to avoid harming cells), among others.
Additionally, enzymes are also used to compose physiological-like fluids that simulate in vivo conditions. These biomimetic environments enable researchers to test and refine their biomaterials under conditions closely resembling the human body, ultimately leading to more effective and clinically relevant regenerative therapies.
The measurement of the substrate’s weight loss and mechanical properties are two common methods to assess the performance of enzymes and to characterize degradation. Table 1 summarizes the differences in some practical aspects between the weight loss technique and the mechanical testing using the ElastoSens™ Bio. Overall, weight loss can lack consistency and sensitivity which can be provided with the mechanical testing.
Table 1: Practical differences between methodologies for the characterization of tissues and organs degradation (weight loss and mechanical measurements with the ElastoSensTM Bio) during the exposure to enzymes.
In this study, the degradation of kidney tissue due to the exposure to collagenase was evaluated through its viscoelastic properties non-destructively measured (during the whole experiment) using the ElastoSens™ Bio. Different concentrations of the enzyme were tested to analyze the consistency and the sensitivity of this method to characterize the degradation of tissues.
MATERIALS AND METHODS
Calf kidney was minced, and 0.3 g was added into the µ-volume sample holder of the ElastoSens™ Bio following the methodology 2 previously described (Figure 1A-B). Collagenase NB4G (Nordmark, Germany) was dissolved in PBS at the concentration of 0.15 U/mL, 0.5 U/mL and 1.5 U/mL. Prior to the first test on the ElastoSens™ Bio (test at t0), the prepared samples were immersed in pure PBS at 37 °C for 20 min. Then, the samples were incubated into 3 mL of the collagenase solutions (n=5 for each collagenase concentration) in a 12-well plate at 37 °C for a total of 1 hour (Figure 1C). Every 15 min, the samples were taken and tested in the ElastoSensTM Bio for 2 min at 37 °C (and re-incubated again after testing). Average results are expressed as mean ± standard deviation. Comparisons among groups were evaluated by one-way ANOVA with post-hoc Tukey test to correct for multiple comparisons. Significance was retained when p < 0.05.
Figure 1: Minced kidney (0.3 g) introduced into the µ-volume sample holder following the methodology 2 (A-B), and sample holders incubated with the collagenase solution in the 12-well plate (C).
RESULTS AND DISCUSSION
Figure 2 illustrates the effect of the collagenase NB4G (0.5 U/mL) on the shear storage modulus (G’) of the kidney samples at each 15 min during the 1-hour incubation. The G’ of the samples gradually decreased during the exposure to collagenase reaching values lower than 40% of its original G’. It is interesting to note the good repeatability of the results especially considering the internal heterogeneity of organs (standard deviations of approximately 10%).
Figure 2: Shear storage modulus (G’) of kidney samples as a function of exposure time to collagenase NB4G (0.5 U/mL) in absolute and relative values.
Figure 3 displays the decrease of G’ in relation to t0 (G’ of the kidney samples before exposure to collagenase) for the three concentrations of collagenase tested. It was found that the increase in enzyme concentration accelerated tissue degradation, showing statistical differences already after 15 min of incubation (p<0.05). Kidney tissue reached 51.8 ± 9.6 %, 39.1 ± 3.4 %, and 24.5 ± 3.4 % of its original G’ after 1-hour incubation with collagenase NB4G at 0.15 U/mL, 0.5 U/mL and 1.5 U/mL, respectively.
Figure 3: Relative shear storage modulus (G’, %) of kidney samples as a function of exposure time to collagenase NB4G at 0.15 U/mL (blue), 0.5 U/mL (turquoise) and 1.5 U/mL (orange).
CONCLUSIONS
Native tissue cut into small pieces were successfully loaded and tested into the µ-volume sample holder of the ElastoSens™ Bio. The use of this holder facilitates an efficient enzyme action by maximizing the sample’s surface area exposed to the enzymatic solution within a 12-well plate. The exposure of kidney samples to collagenase NB4G led to a gradual degradation precisely measured by the ElastoSens™ Bio in terms of viscoelastic properties.
Impact & Use
- Developing an easy and consistent protocol for precisely measuring the degradation of tissues and biomaterials caused by exposure to degrading solutions.
- Comparing the performance of different enzymes and extraction buffers on the target tissues or biomaterials.
- Optimizing the dosage of enzymes and other components to achieve desired degradation time and extent, enhancing cell extraction yields.
- Identifying cost-effective enzyme candidates, dosages, and durations to reduce the overall degradation process cost.
- Ensuring the batch-to-batch consistency and efficacy of an enzyme.
- Optimizing the performance of enzymes and extraction buffers to specific tissues and requirements.
ElastoSens™ Bio
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