Isolation and culture of primary alveolar epithelial cells in vitro

In vitro primary alveolar epithelial cells (Primary Alveolar Epithelial Cell s) Isolation and culture of
1, completely dirty blood residue lung tissue:
2, digestion of lung tissue:
Common cell digestive enzymes: trypsin, elastase, collagenase.
Transbronchial infusion of the enzyme into the intact lung, or the lung tissue is cut into the digestive juice for digestion. The former is more effective than the latter.
3, separation of purified cells:
The main methods for the separation and purification of primary alveolar epithelial cells:
(1) Density gradient centrifugation:
The principle of centrifugation of cells by density gradient is that the sedimentation coefficients of different cells are different, and the positions of cells in density gradient centrifugation are also different. Alveolar type II epithelial cells were isolated by ficoll or percoll discontinuous gradient centrifugation.
(2) Filter separation method:
Alveolar type II epithelial cells are about 10 mm smaller than macrophages (about 25 mm) and type I cells (50 mm-100 mm). Large tissue fragments are removed by filtration with a 150 mm pore size filter, and the filter is removed from the 30 mm to 40 mm pore size. Type II cells and some macrophages were finely filtered using a 15 mm filter. Separation with a membrane is simple and it can be used in combination with other separation methods to increase purity.
(3) Flow cytometry method:
Screening was performed with fluorescent material markers based on differences in fluorescence between different cells. Alveolar epithelial cells are rich in lipids and labeled with lipophilic fluorescein to obtain highly pure cells. Flow-through technology separates cells with very low yields but is extremely pure. This technology will be more attractive in the future.
(4) Immunoadhesion method:
The culture plate was coated with IgG, and the alveolar epithelial cell suspension was placed on the plate. Macrophages and most other non-alveolar epithelial cells bound to IgG due to the receptor containing the Fc fragment, adsorbed to the plate, and adhered to the alveoli. The epithelial cells are washed out, which is simple and easy to remove, and can remove most macrophages, effectively improving the purity of alveolar epithelial cells.
(5) Adhesion selection method:
Principle of adherence selection: alveolar epithelial cells have different time of adherence, and this characteristic is screened when cultured with alveolar epithelial cells.
In summary, according to the ultimate goal of alveolar epithelial cell culture, several methods can achieve the desired results.
For example: (1) first filter with a filter; (2) use IgG coating method.

Ventilator block diagram
One. Main mechanical ventilation modes
(1) Intermittent Positive Pressure Ventilation (IPPV): positive pressure in the inspiratory phase and zero pressure in the expiratory phase. 1. Working principle: The ventilator generates positive pressure in the inspiratory phase and presses the gas into the lungs. After the pressure rises to a certain level or the inhaled volume reaches a certain level, the ventilator stops supplying air, the exhalation valve opens, and the patient's thorax Passive collapse of the lungs and exhalation. 2. Clinical application: Various patients with respiratory failure mainly based on ventilation function, such as COPD.
(2) Intermittent positive and negative pressure ventilation (IPNPV): the inspiratory phase is positive pressure and the expiratory phase is negative pressure. 1. How it works: The ventilator works both in the inspiratory and exhaled phases. 2. Clinical application: Expiratory negative pressure can cause alveolar collapse and cause iatrogenic atelectasis.
(3) Continuous positive pressure airway ventilation (CPAP): Refers to the patient's spontaneous breathing and artificial positive airway pressure during the entire respiratory cycle. 1. Working principle: Inspiratory phase gives continuous positive pressure air flow, and exhalation phase also gives a certain resistance, so that the airway pressure of inhalation and exhalation phases are higher than atmospheric pressure. 2. Advantages: The continuous positive pressure airflow during inhalation is greater than the inspiratory airflow, which saves the patient's inhalation effort, increases FRC, and prevents the collapse of the airway and alveoli. Can be used for exercise before going offline. 3. Disadvantages: great interference to circulation, large pressure injury of lung tissue.
(4) Intermittent command ventilation and synchronized intermittent command ventilation (IMV / SIMV) IMV: There is no synchronization device, the ventilator air supply does not require the patient's spontaneous breathing trigger, and the time of each air supply in the breathing cycle is not constant. 2. SIMV: There is a synchronization device. The ventilator gives the patient a commanded breath according to the pre-designed breathing parameters every minute. The patient can breathe spontaneously without being affected by the ventilator. 3. Advantages: It exerts its ability to regulate breathing while offline; it has less influence on circulation and lungs than IPPV; it reduces the use of shock medicine to a certain extent. 4. Application: It is generally considered to be used when off-line. When R <5 times / min, it still maintains a good oxygenation state. You can consider off-line. Generally, PSV is added to avoid respiratory muscle fatigue.
(5) Mandatory ventilation per minute (MMV) When spontaneous breathing> preset minute ventilation, the ventilator does not command ventilation, but only provides a continuous positive pressure. 2. When spontaneous breathing is less than the preset minute ventilation volume, the ventilator performs command ventilation to increase the minute ventilation volume to reach the preset level.
(6) Pressure Support Ventilation (PSV) Definition: Under the prerequisite of spontaneous breathing, each inhalation receives a certain level of pressure support, increasing the patient's inhalation depth and inhalation volume. 2. How it works: The inspiratory pressure begins with the patient's inspiratory action, and ends when the inspiratory flow rate decreases to a certain level or the patient attempts to exhale hard. Compared with IPPV, the pressure it supports is constant, and it is adjusted by the feedback of the inspiratory flow rate. Compared with SIMV, it can get pressure support for each inhalation, but the level of support can be set according to different needs. 3. Application: SIMV + PSV: used for preparation before off-line, can reduce breathing work and oxygen consumption Indications: Exercise the ventilator; prepare before going offline; the ventilator is weak due to various reasons; severe flail chest causes abnormal breathing. 5. Note: Generally not used alone, it will produce insufficient or excessive ventilation.
(7) Volume Supported Ventilation (VSV): Each breath is triggered by the patient's spontaneous breathing. The patient can also breathe without any support and can reach the expected TV and MV levels. The ventilator will allow the patient to be truly autonomous Breathing also applies to preparations before going offline.
(8) Capacity control of pressure regulation
(IX) Biphasic or bilevel positive pressure ventilation How it works: P1 is equivalent to inspiratory pressure, P2 is equivalent to breathing pressure, T1 is equivalent to inspiratory time, and T2 is equivalent to exhalation time. 2. Clinical application: (1) When P1 = inspiratory pressure, T1 = inspiratory time, P2 = 0 or PEEP, T2 = expiratory time, which is equivalent to IPPV. (2) When P1 = PEEP, T1 = infinity, P2 = 0, T2 = O, which is equivalent to CPAP. (3) When P1 = inspiratory pressure, T1 = inspiratory time, P2-0 or PEEP, T2 = desired controlled inhalation cycle, equivalent to SIMV.


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