Viability testing of homograft valves using methyl thiazol tetrazolium assay.SS Shetty, SS Kaushik, MM Mojamdar, AA Gogate, AP Chaukar
Department of Microbiology, Lokmanya Tilak Municipal Medical College, Sion, Mumbai.
Viability status before and after treatment with antibiotics was investigated in a total of 104 homograft valves using MTT (3-[4, 5-dimethyl thiazol-2-y1]-2, 5-diphenyl tetrazolium bromide). The valves with warm ischaemic time above 11 hours, were found to be non-viable. Increase in storage time directly decreases cell viability. Methyl thiazol tetrazolium (MTT) assay can be used as a reliable, simple, rapid and economic method for assessing the viability status based on mitochondrial respiration even for homograft valves. Basal media with and without nutrients i.e., DMEM and Hanks BSS showed no difference in viability of the cells.
Keywords: Adolescent, Adult, Antibiotics, Cause of Death, Cell Survival, Cryopreservation, methods,Dyes, Female, Heart Valves, transplantation,Human, Isotonic Solutions, Male, Middle Age, Tetrazolium Salts, Thiazoles, Time Factors, Transplantation, Homologous,
Homograft heart valves have different sources of procurement and hence there is considerable variation in the viability status of these valves. We procure the homo-grafts from routine autopsy material which is the only source in our hospital. Yankah et al claimed that there was evidence of cellular viability of valves upto 30 hours after death. The post mortem valves almost always are contaminated and require sterilisation which also can affect viability, lonescu et al stated that donor fibroblasts are the only cells capable of producing collagen in the tissue. If they remain viable, it is believed that they would provide the best conditions for post transplant biology and long-term durability.
It viability is considered important for durability it would be necessary to know the viability status of valves at procurement and after sterilisation. This will enable us to find out suitable methods for preservation of viability.
The main purpose of this study was to assess the presence of viable cells in the valves obtained from post-mortem under our conditions. The viability if present at zero hours (i.e., immediately after procurement) has to be assessed so as to conclude the effects on viability after necessary sterilisation procedures.
If sterile valves thus obtained show sizable viability then methods to preserve it cat be probed into Cryopreservation has wrongly become synonymous with viability. Only viable valves need to be preserved by cryopreservation.
A total of 104 homograft valves were studied from donors aged between 15 and 60 years. After procurement of the valve from the mortuary of Lokmanya Tilak Municipal Medical Hospital, Sion, Mumbai, India, the information on the age, sex, cause of death, blood group, details of blood transfusion, if any, ambient temperature to which the body was exposed after death and before autopsy were noted. Valves were randomly allocated to different groups:
Group I - Valves stored in DMEM* without antibiotics
Group II - Valves stored in DMEM with antibiotics
Group III- Valves stored in Hanks** BSS with antibiotics.
Blood samples from these donors were screened for HIV, Hepatitis B surface antigen and VDRL and only those found negative were further investigated.
Hearts were, collected in normal sterile saline within 30 hours after death. They were washed in running tap water for half an hour. This was followed by dissection and separation of the heart valves in the laminar airflow. The valves were thoroughly washed with normal saline to remove any adhering blood clots etc. and were allocated into three groups as above. From each heart both aortic and pulmonary valves were taken. From each valve either cusp or conduit wall pieces or both were taken for assessing viability.
All the valves in Group I were further studied for viability at zero hours, 24 hours and 48 hours. The valves from Group II and Group II were studied for viability at zero hours, 24 and 48 hours respectively.
The DMEM / Hanks BSS had antibiotic composition as follows:
Benzyl penicillin - 1 Lakh units
Streptomycin - 0.75 grams
Hanks BSS */DMEM** - 100 millilitres
Incubate at 37 deg. C, for 2 hours
Wash with plain sterile Hanks BSS/DMEM
Cloxacillin - 250 milligrams
Gentarnicin - 80 milligrams
Hanks BSS */DMEM * - 100 millilitres
Incubate at 37 deg. C. for 2 hours and then store at 4 deg. C
* DMEM - Dulbeccos Modification of Eagles Media
** Hanks BSS - Hanks Balanced Salt Solution
The valves in DMEM / Hanks viz Group II and Group II after antibiotic treatment were stored at 4 deg. C whereas the Group I valves were stored directly at 4 deg.C.
Mitochondrial succinate dehydrogenase dependent reduction of 3 - (4,5- dimethylthiazol - 2-y1) -2,5- diphenyl tetrazolium bromide (MTT) to its purple coloured formazan crystals was used to obtain a measure of the viability status of the cusps and wall of conduits of the aortic and pulmonary valves.
Modification of Mosmarin's method was used. Briefly the pieces of cusps and pieces of wall of the conduits of approximately 1 MM were weighed in a sterile test tube. To this was added 500 micro-litres of DMEM and 50 micro-litres of 1 mg/ml MTT (Sigma) solution prepared in phosphate buffered saline (PBS) pH 7.2 and incubated at 37 deg C. for 4 hours. The formazan crystals formed were dissolved in 1 ml M 0.02N acid iso-propanol (E Merck) (1 ml HCI: 99 ml Isopropanol)and read in a spectrophoto meter, Absorbance of converted dye was measured at 570 nm with background subtraction at 630 nm. Since this method depends on active mitochondrial dehydrogenases enzymes of viable cells for conversion of the dye, it has the added advantage of being carried out with no interference from the contaminating microorganisms.
The data measured on continuous scale were statistically analysed using Student's t-test and categorical data were subjected to Chi-square test. The differences for which p-valves remained below 0.05 were labelled as statistically significant test.
For determination of cut-off value, ten negative controls were used. For negative control the tissue pieces were boiled at 100 deg. C. for half an hour and then MITT assay was performed. To get the range of viability, seven viable samples from the operated cases were collected. This formed the known positive control. The cut-off value was determined as follows: Cut-off value = Mean of ten negative controls + 3 Standard deviation = 0.168 + 3 (0.01498) = 0.212
Any optical density for 100 milligram wet weight above the cut-off value was considered as positive and values below this were considered negative. The positive control mean was 1.082.
With each batch of the test sample, a negative control was run for quality control.
The distribution of hearts in three groups and their baseline details are presented in [Table - 1].
In Group I, 24 valves were tested for viability. On the basis of analysis it was found that the valves harvested within 11 hours of warm ischaemic time (WIT) showed viability.
Only one valve showed retention of viability on procurement with warm ischaemic time more than 11 hours and this viability was not retained thereafter. In this study both cusp and pieces of conduit wall of the valves were taken for viability testing.
In Group I, the retention of viability between cusp and conduit of subgroup A as well as subgroup B failed to reach statistical significance.
18 cusps and 18 pieces of wall of conduits with WIT less than 11 hours were studied, of which 13, five and four cusps were viable at zero, 24 and 48 hours respectively and 12, five and four pieces of wall of conduit were viable at zero, 24 and 48 hours respectively. Six cusps and six pieces of conduit wall with WIT greater than 11 hours were studied and only one cusp and one piece of conduit were viable at zero hours and non viable thereafter, the WIT of this valve was 13 hours and 35 minutes. In Group I the difference between subgroups A and B at zero hours was statistically significant (p < 0.05). At the end of 24 and 48 hours, however failed to reach statistical significance [Table - 2].
In Group II, 24 valves were used for viability testing and these were stored in DMEM. After harvesting, viability testing was done at zero, four, 24 and 48 hours respectively followed antibiotic treatment. In this study only pieces of conduit wall were taken. Out of the twelve pieces of conduit wall with WIT less than 11 hours all the twelve were viable at zero and four hours. Six and four were viable at the end of 24 and 48 hours respectively. Out of the twelve conduits with WIT more than 11 hours only one valve was viable at zero hours and nonviable thereafter. WIT of this valve was 13 hours 40 minutes. The difference than 11 hours only one valve between the subgroups A and B of Group II both at zero and four hours was statistically significant (p < 0.05). At the end of 24 and 48 hours, however the differences failed to reach statistical significance [Table - 3].
In the Group III, 56 valves were studied. Viability testing of valves stored in Hanks BSS was done at zero, four, 24 and 48 hours. Only conduit wall pieces were taken for viability testing and of the 12 conduit wall pieces with WIT less than 11 hours, 11 were viable at zero and at the end of four hours and two each were viable at the end of 24 and 48 hours. Out of 44 conduit wall pieces with WIT greater than 11 hours, two showed viability at zero hours and in only one case there was retention of viability whose WIT was 17 hours 35 minutes. The difference between the subgroups of Group III both at zero and four hours were statistically significant (p<0.05). At the end of 24 and 48 hours, however the difference failed to reach statistical significance [Table - 4].
This study has been conducted to know whether the valves obtained at autopsy are viable under our circumstances. If they are viable, cryopreservation (Liquid - vapour phase of liquid nitrogen i.e,-1143 to -196 deg. C) technique can be used for maintaining this viability.
There has been a long felt need to develop a methodology to assess the viability status of the homograft valves as they are contaminated under our circumstances. In this study, we have used the system first described by Mosmann wherein the tetraolium salt MTT is converted to its coloured formazan by the mitochondrial enzymes of viable cells and this assay has also been modified by Slaclwoski. As bacteria do not have mitochondria, this method only detected the viability of valve tissue. The outcome of this assay are thus not affected by bacterial contamination, while those of other methods like glucose, thymidine uptake etc would be interfered with in case of bacterial contamination.
Bank et al stated that the endothelial cells from human heart valves are damaged during procurement or subsequent processing. The viability detected by MTT assay are probably the fibroblasts cells, but the same has not been proved.
Further in this study it was found that wall pieces of conduit and cusps do not differ significantly References in their viability status. Therefore the former can be used for viability assays and the results extrapolated to homograft valves safely. So such valves can be used for transplantation, thereby avoiding wastage of valves.
Our data showed that warm ischaemic time played an important role with respect to the viability of valves. Our study indicates that under our circumstances WIT more than 11 hours shows no viability. The warm ischaemic time is an important determinant of viability. The donor is exposed to ambient temperature (32-40 deg. C) for two hours after death as per existing rules. After this period only the body is preserved in cold storage till autopsy.
Cell viability using flow cytometry was studied by Niwaya et al and they stated that cell viability of the cryopreserved allograft valves was well preserved with a WIT < 520 minutes (8.7 hours). Crescenzo et al in an electron microscopic study observed that heart valves harvested within a warm ischaemic time of 12 hours contain a population of matrix cells, a majority of which are morphologically unaltered and thus presumably will be viable after transplantation. In contrast those with warm ischaemic time of more than 12 hours had matrix cells that were irreversibly injured with mitochondrial flocculency. The above mentioned results are in close agreement with the findings of the present study and thus suggest that the MTT assay can be used as a rapid, economic and qualitative method and can be performed in a busy cardiovascular laboratory.
There was no difference in the maintenance of viability even though additional nutrients were provided using DMEM. This concurred with the data of Chalcroft et al. Cell viability decreases with storage time at 4 deg. C due to effect of low temperature on their metabolic activities. Hu et al have also found that storage at 4 deg. C alone decreases the viability by 50%, 60% and 90% in 12, 24 and 72 hours respectively.
In conclusion, this study states that under our present circumstances and with WIT above 11 hours the homograft valves are non viable. As most of the homograft valves procured are non-viable, cryopreservation is not the method of choice for us.
Mr. Suresh Bowalekar, Chartered Statistician, Deputy General Manager, Statistics & Clinical trial, Wockhardt Ltd., India
[Table - 1], [Table - 2], [Table - 3], [Table - 4]