A human ex vivo model of haemostasis: A replacement for rodent tail bleeding assays
Lead Research Organisation:
Manchester Metropolitan University
Department Name: School of Healthcare Science
Abstract
Haemostasis is the important biological process that controls blood clotting. It is a complicated process involving the blood vessel wall, cells in the blood and proteins circulating in plasma. Understanding how haemostasis is regulated is crucial, as when it goes wrong it can lead to life threatening bleeding or clotting.
Currently the main way to investigate factors which affect haemostasis is by using the tail bleeding model. This involves cutting off the end of a rodents tail and timing how long it takes to stop bleeding. Different drugs and treatments can then be tested on the animals to see if they change the bleeding time. These experiments are very variable and therefore require lots of animals. They are also not a very good representation of haemostasis in humans as there are many biological differences between rodents and humans.
The aim of this study is to develop an alternative haemostasis model that uses human blood and human blood vessels to replace the tail bleeding model. To do this we will take human blood vessels from placentas, which are usually discarded following the birth of a baby. We will flush out existing blood from the vessel and flow human donor blood through it. When blood is passing through the vessel, we will use a small needle to puncture the blood vessel wall causing bleeding. This will stimulate the formation of a blood clot and we will time how long it take to stop bleeding.
As well as measuring bleeding time in our human haemostasis model, we will also use high power microscopes to look in detail at what the blood clots are made up of. This will tell us more information about the process of haemostasis and how it is controlled.
To enable other scientists to use our model and have a greater impact reducing the number of animals used in research, they need access to human placental vessels. We will therefore test different ways of storing the blood vessels, so that vessels can be stored for longer and scientists can use the model without access to fresh placentas.
In summary, this study will provide a new way to measure haemostasis that does not use animals. It will also produce more reliable results and provide more detailed information about how haemostasis is controlled. The use of human blood and human blood vessels also means that the results are more relevant to human haemostasis than the current tail bleeding model.
Currently the main way to investigate factors which affect haemostasis is by using the tail bleeding model. This involves cutting off the end of a rodents tail and timing how long it takes to stop bleeding. Different drugs and treatments can then be tested on the animals to see if they change the bleeding time. These experiments are very variable and therefore require lots of animals. They are also not a very good representation of haemostasis in humans as there are many biological differences between rodents and humans.
The aim of this study is to develop an alternative haemostasis model that uses human blood and human blood vessels to replace the tail bleeding model. To do this we will take human blood vessels from placentas, which are usually discarded following the birth of a baby. We will flush out existing blood from the vessel and flow human donor blood through it. When blood is passing through the vessel, we will use a small needle to puncture the blood vessel wall causing bleeding. This will stimulate the formation of a blood clot and we will time how long it take to stop bleeding.
As well as measuring bleeding time in our human haemostasis model, we will also use high power microscopes to look in detail at what the blood clots are made up of. This will tell us more information about the process of haemostasis and how it is controlled.
To enable other scientists to use our model and have a greater impact reducing the number of animals used in research, they need access to human placental vessels. We will therefore test different ways of storing the blood vessels, so that vessels can be stored for longer and scientists can use the model without access to fresh placentas.
In summary, this study will provide a new way to measure haemostasis that does not use animals. It will also produce more reliable results and provide more detailed information about how haemostasis is controlled. The use of human blood and human blood vessels also means that the results are more relevant to human haemostasis than the current tail bleeding model.
Technical Summary
Haemostasis is the biological process which maintains blood flow around the body and prevents bleeding following injury through the formation of a haemostatic plug. It is a complex and tightly regulated process involving the blood vessel wall, platelets, and soluble factors in plasma. Loss of regulation can be catastrophic resulting in life threatening bleeding or clotting.
To understand the complex cellular and molecular mechanisms which underpin haemostasis, all principal components are required in a single model. The current gold standard is the tail bleeding assay in rodents. This involves amputation of the end of the tail and recording the time taken to stop bleeding. The assay is poorly standardized, extremely variable, requiring 10-20 animals per experimental group, and is limited by fundamental species differences.
The aim of this study is to develop a human ex vivo model of haemostasis as an alternative to the tail bleeding assay. The model will comprise of human blood perfused through a human placental blood vessel. The blood vessel wall will be injured by a puncture wound of a specific diameter and bleeding time recorded as the time taken to cease bleeding. This will be coupled with fluorescent imaging and scanning electron microscopy, enabling clot structure, composition, and morphology to be analysed. The model will be validated by comparing bleeding times between the ex vivo model and published data using the tail bleeding assay in the presence of drugs known to alter haemostasis.
To ensure that the ex vivo model can be widely adopted we will test a range of tissue preservation methods including cold storage and cryopreservation to facilitate tissue accessibility and potential future commercialization of the model. Successful delivery of this project will provide a replacement for the archaic tail bleeding model with a more reproducible, informative, and relevant model of human haemostasis.
To understand the complex cellular and molecular mechanisms which underpin haemostasis, all principal components are required in a single model. The current gold standard is the tail bleeding assay in rodents. This involves amputation of the end of the tail and recording the time taken to stop bleeding. The assay is poorly standardized, extremely variable, requiring 10-20 animals per experimental group, and is limited by fundamental species differences.
The aim of this study is to develop a human ex vivo model of haemostasis as an alternative to the tail bleeding assay. The model will comprise of human blood perfused through a human placental blood vessel. The blood vessel wall will be injured by a puncture wound of a specific diameter and bleeding time recorded as the time taken to cease bleeding. This will be coupled with fluorescent imaging and scanning electron microscopy, enabling clot structure, composition, and morphology to be analysed. The model will be validated by comparing bleeding times between the ex vivo model and published data using the tail bleeding assay in the presence of drugs known to alter haemostasis.
To ensure that the ex vivo model can be widely adopted we will test a range of tissue preservation methods including cold storage and cryopreservation to facilitate tissue accessibility and potential future commercialization of the model. Successful delivery of this project will provide a replacement for the archaic tail bleeding model with a more reproducible, informative, and relevant model of human haemostasis.
